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1.
Nat Genet ; 9(2): 184-90, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7719347

RESUMO

The largest known gene is the human dystrophin gene, which has 79 exons spanning at least 2,300 kilobases (kb). Transcript accumulation was monitored from four regions of the gene following induction of expression in muscle cell cultures. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) results indicate that approximately 12 h are required for transcription of 1,770 kb (at an average elongation rate of 2.4 kb min-1), extrapolating to a transcription time of 16 h for the complete gene. Accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5' end before transcription is complete providing strong evidence for cotranscriptional splicing. The rate of transcript accumulation was reduced at the 3' end of the gene relative to the 5' end, perhaps due to premature termination of transcription complexes.


Assuntos
Distrofina/genética , Transcrição Gênica , Animais , Sequência de Bases , Diferenciação Celular , DNA Recombinante , Éxons , Expressão Gênica , Humanos , Dados de Sequência Molecular , Músculos/citologia , Reação em Cadeia da Polimerase/métodos , Fatores de Tempo
2.
Science ; 242(4879): 755-9, 1988 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-3055295

RESUMO

Duchenne muscular dystrophy (DMD) and its less severe form Becker muscular dystrophy (BMD) are allelic disorders. It has been suggested that in the mutations involving BMD, the translational reading frame of messenger RNA is maintained and a smaller, though partially functional, protein is produced. In order to test this, the exon-intron boundaries of the first ten exons of the DMD gene were determined, and 29 patients were analyzed. In a number of BMD patients (mild and severe BMD), the reading frame of messenger RNA was not maintained. On the basis of these findings, a model for reinitiation from an internal start codon is suggested.


Assuntos
Proteínas Musculares/genética , Distrofias Musculares/genética , Cromossomo X , Sequência de Bases , Southern Blotting , Deleção Cromossômica , Sondas de DNA , Distrofina , Éxons , Genes , Humanos , Mutação , Fenótipo
3.
Mol Cell Biol ; 10(1): 193-205, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2403634

RESUMO

Duchenne muscular dystrophy (DMD) gene transcripts are most abundant in normal skeletal and cardiac muscle and accumulate as normal myoblasts differentiate into multinucleated myotubes. In this report we describe our initial studies aimed at defining the cis-acting sequences and trans-acting factors involved in the myogenic regulation of DMD gene transcription. A cosmid clone containing the first exon of the DMD gene has been isolated, and sequences lying upstream of exon 1 were analyzed for homologies to other muscle-specific gene promoters and for their ability to direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs. The results indicate that the transcriptional start site for this gene lies 37 base pairs (bp) upstream of the 5' end of the published cDNA sequence and that 850 bp of upstream sequence can direct CAT gene expression in a muscle-specific manner. Sequence analysis indicates that in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. Our observation that only 149 bp of upstream sequence is required for muscle-specific expression of a chimeric CAT gene construct further implicates the CArG and myocyte-specific enhancer-binding nuclear factor 1 binding homologies as important domains in the regulation of this gene. On the other hand, the unique profile of myogenic cell line-specific induction displayed by our DMD promoter-CAT gene constructs suggests that other as yet undefined cis-acting sequences and/or trans-acting factors may also be involved.


Assuntos
Proteínas Musculares/genética , Músculos/fisiologia , Distrofias Musculares/genética , Regiões Promotoras Genéticas , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Clonagem Molecular , Análise Mutacional de DNA , Distrofina , Amplificação de Genes , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Músculos/citologia , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transfecção
4.
Gene ; 200(1-2): 173-6, 1997 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-9373152

RESUMO

A region of 744 basepairs (bp) upstream of the muscular dystrophin promoter (UMDP) was amplified by inverse-polymerase chain reaction (PCR), cloned and sequenced. Analysis of this sequence for the presence of putative transcriptional control elements identified several similarities with known cis-acting sequence motifs including two MyoD and two Ap1 motifs. One of these Ap1 motifs was found to be completely conserved within an otherwise highly variable region among five primate species. Complete homology to a human fetal brain expressed sequence tag (EST) was also observed over 201 bp at the 5' end of the UMDP region. Northern blot analysis using a radiolabelled EST probe identified a 1 kb mRNA expressed in human placenta and at lower levels in the heart. These results raise the possibility that additional transcriptional regulatory elements are located upstream of the core muscle promoter, and provide the first evidence for the existence of a gene that overlaps the human dystrophin gene.


Assuntos
Encéfalo/metabolismo , Distrofina/genética , Evolução Molecular , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Cerebelo/metabolismo , Sequência Conservada , Éxons , Feto , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Células de Purkinje/metabolismo , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Sitios de Sequências Rotuladas
5.
FEBS Lett ; 441(2): 337-41, 1998 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-9883911

RESUMO

The Dp71 dystrophin isoform has recently been shown to localize to actin filament bundles in early myogenesis. We have identified an actin binding motif within Dp71 that is not found in other dystrophin isoforms. Actin overlay assays and transfection of COS-7 cells with fusion proteins of wild type and mutated Flag epitope-tagged Dp71 demonstrate that this motif is necessary and sufficient to direct localization of Dp71 to actin stress fibers. Furthermore, this localization is independent of alternative splicing which alters the C-terminus of the protein. The identification of an actin binding site suggests Dp71 may function to anchor membrane receptors to the cytoskeleton.


Assuntos
Actinas/metabolismo , Distrofina/análogos & derivados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Citoesqueleto/metabolismo , Distrofina/química , Distrofina/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
6.
Cancer Gene Ther ; 6(2): 179-90, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10195885

RESUMO

The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents.


Assuntos
Neoplasias da Mama/terapia , Ganciclovir/uso terapêutico , Terapia Genética , Simplexvirus/genética , Timidina Quinase/genética , Laranja de Acridina/metabolismo , Adenoviridae/metabolismo , Apoptose , Northern Blotting , Western Blotting , Sobrevivência Celular , Fragmentação do DNA , Etídio/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sensibilidade e Especificidade , Timidina Quinase/administração & dosagem , Fatores de Tempo , Transdução Genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
7.
Cancer Gene Ther ; 7(12): 1566-74, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11228535

RESUMO

Targeting therapeutic gene expression to tumor cells represents a major challenge for cancer gene therapy. The strong transcriptional response exhibited by heat shock genes, along with the beneficial therapeutic effects of hyperthermia have led us to develop a heat-directed gene-targeting strategy for cancer treatment. Heat shock gene expression is mediated in large part by the interaction of heat shock factor 1 with specific binding sites (heat shock elements; HSE) found in the promoters of heat-inducible genes. Here we present a quantitative analysis of heat-inducible gene expression mediated by the wild-type hsp70b gene promoter, as well as a modified hsp70b promoter containing additional HSE sequences. Beta-galactosidase (beta-gal) expression was induced between 50- and 800-fold in a panel of human breast cancer cell lines infected with an adenoviral vector containing the wild-type hsp70b promoter (Ad.70b.betag) following treatment at 43 degrees C for 30 minutes. Infection with an adenoviral vector containing the modified hsp70b promoter (Ad.HSE.70b.betag) resulted in a 200- to 950-fold increase in beta-gal expression under the same conditions, and also provided a 1-2 degrees C decrease in the threshold of activation. Significant increases in the heat responsiveness of the Ad.HSE.70b.betag construct were observed in five of six tumor cell lines tested, as well as under thermotolerant conditions. Finally, we demonstrate that localized heating of a HeLa cell xenograft can effectively target beta-gal gene expression following intratumoral injection of Ad.70b.betag. Adenoviral vectors incorporating heat-inducible therapeutic genes may provide useful adjuncts for clinical hyperthermia.


Assuntos
Adenoviridae/genética , Marcação de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos , Hipertermia Induzida , Células Tumorais Cultivadas/metabolismo , Animais , Feminino , Expressão Gênica , Genes Reporter , Proteínas de Choque Térmico HSP70/genética , Humanos , Interleucina-12/genética , Camundongos , Camundongos SCID , Transfecção , beta-Galactosidase/metabolismo
8.
Int J Radiat Oncol Biol Phys ; 42(2): 331-4, 1998 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9788412

RESUMO

PURPOSE: To determine, retrospectively, the status of the bp 609 mutation in the DT-diaphorase gene in anal canal carcinoma patients who have undergone radical radiotherapy with concurrent 5-fluorouracil (5-FU) and mitomycin C (MMC), to determine the relationship of the mutant form of the gene to treatment outcomes. METHODS AND MATERIALS: Paraffin blocks of pretreatment tumor biopsies were obtained on 49 patients who underwent treatment with curative intent using radiation, infusional 5-FU and bolus MMC from January 1991 to December 1993. DNA was extracted and subjected to polymerase chain reaction (PCR) analysis using primers that encompassed the bp 609 C to T mutation. Restriction endonuclease cleavage with Hinf 1 and gel electrophoresis were used to determine the polymorphism status of each patient. RESULTS: DNA of 46 patients was successfully amplified. The 46 patients were distributed as follows: 26 (56.5%) C/C-homozygous wildtype, 18 (39%) T/C-heterozygous, and 2 (4.5%) T/T-homozygous mutant. Eleven of 46 patients had suffered treatment failure. The status of the bp 609 polymorphism in this group was 5 (45.5%) C/C, 5 (45.5%) C/T, and 1 (9%) T/T. CONCLUSION: In this series, there was not an overrepresentation of the mutant allele in patients with treatment failure, suggesting that the bp 609 alteration is not a strong determinant of treatment outcome.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Ânus/tratamento farmacológico , Neoplasias do Ânus/radioterapia , NAD(P)H Desidrogenase (Quinona)/genética , Proteínas de Neoplasias/genética , Neoplasias do Ânus/enzimologia , Neoplasias do Ânus/genética , Terapia Combinada , Fluoruracila/administração & dosagem , Humanos , Mitomicina/administração & dosagem , Polimorfismo Genético , Estudos Retrospectivos , Resultado do Tratamento
9.
Neuromuscul Disord ; 10(3): 187-93, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10734266

RESUMO

Transcripts encoding the 70-75 kDa C-terminal protein product of the dystrophin gene (Dp71) are alternatively spliced to generate multiple protein products in a number of adult human tissues. In this report, reverse transcriptase-polymerase chain reaction was used to clone and characterize a subpopulation of truncated Dp71 transcripts in adult human brain tissue which did not contain exons 71-74, resulting in an in-frame deletion of 330 bp encoding the syntrophin-binding domain. These truncated Dp71 transcripts are also alternatively spliced for exon 78. Immunoblot analysis, using dystrophin-specific C-terminal antibodies directed against epitopes in either exon 77 (MANDRA1), or 78 (1461), identified full-length dystrophin, Dp140 and Dp71, in total protein lysates from adult human brain tissue. In addition, a minor immunoreactive protein of approximately 58 kDa was also identified (designated Dp71 big up tri, open(110)). The observation that a monoclonal antibody directed against epitopes within exons 73-74 (MANEX7374A) failed to detect this 58 kDa protein provides definitive evidence that Dp71 big up tri, open(110) is derived from Dp71 transcripts deleted for the syntrophin-binding domain. These results, as well as previous findings, demonstrate that alternative splicing of Dp71 in the human brain generates a variety of mRNA transcripts encoding distinct protein variants of Dp71, and further supports the use of exon-specific antibodies in characterizing these variants. The presence of these Dp71 protein variants in brain tissue points to their interaction with various cellular proteins and their involvement in different cellular functions.


Assuntos
Processamento Alternativo/genética , Química Encefálica/genética , Distrofina/análogos & derivados , Distrofia Muscular de Duchenne/genética , Clonagem Molecular , Distrofina/genética , Regulação da Expressão Gênica , Humanos , Immunoblotting , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Transcrição Gênica
10.
Radiother Oncol ; 61(3): 309-12, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11731001

RESUMO

BACKGROUND: One major challenge to human cancer gene therapy, is efficient delivery of the gene-vector complex. METHODS AND RESULTS: Using two distinct human nasopharyngeal carcinoma (NPC) models, we demonstrate that intra-tumoural (IT) administration of adenoviral-mediated wild-type p53 gene therapy (Ad-p53) caused no greater inhibition of tumour growth as compared to ionizing radiation (XRT) alone. Detailed histologic examination of tumour sections demonstrated that <15% of tumour cells were transduced by IT adv-beta-gal. CONCLUSIONS: This report underscores the importance of developing gene transfer vectors, which can provide therapeutic levels of transgene expression efficiently in solid tumours.


Assuntos
Adenoviridae , Genes p53 , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias Nasofaríngeas/terapia , Adenoviridae/genética , Animais , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Knockout , Neoplasias Nasofaríngeas/radioterapia , Transplante Heterólogo
11.
Mutat Res ; 406(2-4): 45-54, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10479721

RESUMO

The determination of the gene expression pattern of single cells has important implications for many areas of cellular and developmental biology including lineage determination, identification of primitive stem cells and temporal gene expression patterns induced by changes in the cellular microenvironment. Global Single Cell Reverse Transcription-Polymerase Chain Reaction (GSC RT-PCR) enables the study of single cell gene expression patterns. Initial observations of significant heterogeneity among single cells derived from a population of cells prompted us to determine how much of this observed heterogeneity was due to the intrinsic variation within the method. In this paper we discuss the sensitivity of GSC RT-PCR for analysis of differences in gene expression between single cells and, in particular, detail the amount of variation generated by the method itself. We found that most of the intrinsic variation in the method occurred in the PCR step. The total variation induced by the method was in the range of 5 fold. While we have determined that there is a five fold methodological variation in GSC RT-PCR, any method which use its components (including generation of cDNAs for microarray analysis) is likely to be affected by such experimental variability, which could limit the interpretation of the resulting data.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase , Análise de Variância , Animais , Extratos Celulares/genética , Linhagem Celular , Sondas de DNA , DNA Complementar/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Nucleosídeo NM23 Difosfato Quinases , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Ribossômicas/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismo
12.
Adv Exp Med Biol ; 280: 107-11; discussion 111-2, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2248131

RESUMO

The studies I've outlined here are obviously at a preliminary stage but do offer some insight into the complexity of DMD gene regulation and do suggest that an understanding of this regulation may have some potential benefit in gene therapy for this disease. The high promoter activity found was unexpected and suggests that in vivo the activity of the endogenous gene may be repressed by elements not present within this region. Of course, other interpretations are possible. The transcripts in vivo may turn over very quickly, or the very large size of the DMD gene may in itself limit the rate of transcription. Alternatively, the gene may be actively transcribed only during the early stages of differentiation. A more detailed analysis of developmental expression and of DNA sequences surrounding exon one is required to address these alternatives, but the possibility for augmenting dystrophin synthesis during myoblast therapy clearly exists. The high level of activity and the tissue and developmental specificity exhibited by the HP2 construct suggest this may be the promoter of choice in future gene therapy experiments. The high degree of specificity shown by this promoter would reduce the need to target gene constructs to muscle cells and would reduce the potential complications of uncontrolled gene expression. Of course, before any of these benefits could be realized much more work must be done both in analysing DMD gene expression and in testing potential gene therapy constructs both in culture and in animal models of this disease.


Assuntos
Distrofias Musculares/genética , Regiões Promotoras Genéticas , Mapeamento Cromossômico , Clonagem Molecular , Expressão Gênica , Terapia Genética , Humanos , Masculino , Distrofias Musculares/terapia
13.
Muscle Nerve ; 9(7): 597-605, 1986 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3762581

RESUMO

Normal and dystrophic hamster myoblasts and fibroblasts were compared for characteristic indicators of growth and differentiation. Clonal analysis of myoblast cultures indicated that 80% of colonies judged to be fusion-competent had differentiated. Dystrophic myoblasts were identical to normal in terms of their morphology, fusion potential (81.4%), and myokinase activity (59.6-49.1 mU/mg at 2-7 days), but displayed a significantly higher plating efficiency (normal: 52.6%; dystrophic: 82.1%), a longer doubling time (normal: 21.7 hours; dystrophic: 33.3 hours), and a lower day-7 creatine kinase activity (normal: 60.7 mU/mg; dystrophic: 41.3 mU/mg). Dystrophic fibroblasts were indistinguishable from normal ones in terms of their morphology, plating efficiency (90.4%), and doubling time (32.5 hours), but displayed a significantly lower day-2 creatine kinase activity (normal 58.3 mU/mg; dystrophic: 35.8 mU/mg) and day-7 myokinase activity (normal: 52.7 mU/mg; dystrophic: 39.9 mU/mg). The results are suggestive of an early and differential expression of the primary defect in dystrophic hamster myoblasts and fibroblasts in culture.


Assuntos
Fibroblastos/fisiologia , Desenvolvimento Muscular , Distrofia Muscular Animal/fisiopatologia , Adenilato Quinase/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Células Clonais , Creatina Quinase/metabolismo , Cricetinae , Mesocricetus , Distrofia Muscular Animal/enzimologia
14.
Br J Cancer ; 77(8): 1236-40, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9579828

RESUMO

DT-diaphorase, a homodimeric flavoenzyme, can provide for a defence mechanism against carcinogenesis mediated by dietary or environmental quinones as well as bioactivate quinone-containing chemotherapeutic drugs. Human cell lines and strains have been identified with very low or undetectable enzymatic activity and a C to T transition at nucleotide 609 of the DT-diaphorase cDNA. This single base change is predicted to result in a proline to serine change in amino acid 187. Human cells homozygous for this base transition fail to exhibit Western blot reactivity for DT-diaphorase, suggesting that this substitution results in protein instability. To directly test whether this base change affects DT-diaphorase enzymatic activity and/or protein stability in vivo, mammalian expression vectors containing DT-diaphorase cDNA with or without the nucleotide 609 base transition were transiently transfected in COS-1 cells. Co-transfection with a human growth hormone expression vector allowed normalization for transfection efficiency. COS-1 transfectants expressing the C to T base change displayed at least a tenfold reduction in DT-diaphorase activity (P < 0.001) and a two- to threefold reduction in protein levels compared with wild-type transfectants. These results are the first to detect the presence of DT-diaphorase protein coded for by the 609 base transition in mammalian cells and confirm its predicted reduced enzymatic activity.


Assuntos
Células COS/enzimologia , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/genética , NAD(P)H Desidrogenase (Quinona)/genética , Mutação Puntual , Transfecção , Animais , Composição de Bases , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Primers do DNA/química , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Polimorfismo Genético
15.
Breast Cancer Res Treat ; 48(3): 273-86, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9598874

RESUMO

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.


Assuntos
Apoptose/efeitos dos fármacos , Terapia Genética , Proteína Supressora de Tumor p53/uso terapêutico , Adenoviridae/genética , Neoplasias da Mama/terapia , Feminino , Técnicas de Transferência de Genes , Humanos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética
16.
Muscle Nerve ; 6(6): 436-41, 1983.
Artigo em Inglês | MEDLINE | ID: mdl-6312308

RESUMO

As part of a study on the implication of elevated Ca2+ levels in the myofibrillar degeneration seen in dystrophic muscle, the content of calmodulin and the activity of Ca2+-activated neutral protease (CANP) have been measured in normal and dystrophic (UM-X7.1) hamsters. Calmodulin levels, expressed as micrograms +/- SEM per gram wet weight were highest in brain (385 +/- 24.7), followed by tongue (93.88 +/- 3.93), heart (42.13 +/- 2.93), and skeletal muscle (31.69 +/- 1.42). No significant increases in calmodulin were observed in the dystrophic tissues thus suggesting that the Ca2+ accumulations observed in dystrophic muscles are unrelated to changes in a calmodulin levels. Because of the complexity of regulation of CANP, a time-dependent study was done using extracts of skeletal, heart, and tongue muscles. Marginal increases in dystrophic CANP were seen in skeletal muscle at all times studied and in the heart and tongue at initial time points only. The data are discussed in terms of rising levels of Ca2+ in muscles of the UM-X7.1 hamster being sufficient to increase CANP activity (without increasing content) to where it causes Z-line dissolution.


Assuntos
Calmodulina/metabolismo , Endopeptidases/metabolismo , Distrofia Muscular Animal/metabolismo , Animais , Encéfalo/metabolismo , Calpaína , Cricetinae , Técnicas In Vitro , Músculos/metabolismo , Miocárdio/metabolismo , Fatores de Tempo
17.
Muscle Nerve ; 10(1): 69-76, 1987 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3561440

RESUMO

Calmodulin levels have been assessed in whole muscle and primary culture extracts in order to examine the relationship between calmodulin and the accumulation of calcium in dystrophic hamster muscle tissues. Significant decreases in both normal and dystrophic skeletal muscle, tongue, and heart calmodulin levels were observed between 2 and 12 weeks of age. Dystrophic values, however, tended to be somewhat higher than normal, especially in 12-week-old skeletal muscle total and soluble extracts (normal 29.7 and 0.6 and dystrophic 117.0 and 3.1 micrograms/g wet weight, respectively). No significant differences were observed in dystrophic myoblast (total 2.22-2.78, soluble 2.85-3.26 micrograms/mg protein) or fibroblast (total 2.64-2.94, soluble 2.54-3.60 micrograms/mg protein) calmodulin levels, except for a significant decrease in dystrophic fibroblast levels (total 1.97, soluble 2.18 micrograms/mg protein) at 7 days in culture. Elevated calmodulin levels in dystrophic muscle are discussed in terms of increases in intracellular Ca2+ concentrations and immature regenerating fibers.


Assuntos
Calmodulina/metabolismo , Músculos/metabolismo , Distrofia Muscular Animal/metabolismo , Animais , Células Cultivadas , Cricetinae , Fibroblastos/metabolismo , Mesocricetus
18.
Nucleic Acids Res ; 25(8): 1618-25, 1997 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-9092671

RESUMO

In previous studies we have described a 5.0 kb Hin dIII fragment downstream of muscle exon 1 that exhibits properties consistent with a muscle-specific transcriptional enhancer. The goal of this study has been to identify the sequence elements responsible for muscle-specific enhancer activity. Functional studies indicated that this enhancer is active in pre- and post-differentiated H9C2(2-1) myoblasts but functions poorly in L6 and C2C12 myotubes. The core enhancer region was delimited to a 195 bp Spe I- Acc I fragment and sequence analysis identified three MEF-1/E box and two MEF-2/AT-rich motifs as potential muscle-specific regulatory domains. EMSA competition and DNase footprinting indicated that sequences within a 30 bp region containing single adjoining MEF-1/E box and MEF-2/AT-rich motifs are target binding sites for trans -acting factors expressed in H9C2(2-1) myotubes but not in L6 or C2C12 myotubes. Site-specific mutations within these motifs resulted in a significant reduction in enhancer activity in H9C2(2-1) myotubes. These results suggest that the mechanisms governing DMD gene expression in muscle are similar to those identified in other muscle-specific genes. However, the myogenic profile of enhancer activity and trans -acting factor binding suggests a more specialized role for this enhancer that is consistent with its potential involvement in dystrophin gene regulation in cardiac muscle.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Distrofina/biossíntese , Distrofina/genética , Elementos Facilitadores Genéticos , Íntrons , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Adenina , Composição de Bases , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Cerebelo/metabolismo , Primers do DNA , Genes Reporter , Humanos , Fatores de Transcrição MEF2 , Dados de Sequência Molecular , Fatores de Regulação Miogênica , Reação em Cadeia da Polimerase , Células de Purkinje/metabolismo , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Timina , Transcrição Gênica
19.
Biochem J ; 227(2): 583-9, 1985 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-4004781

RESUMO

L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.


Assuntos
Lipase/metabolismo , Lisossomos/enzimologia , Músculos/enzimologia , Fosfatase Ácida/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Detergentes/farmacologia , Músculos/citologia , Músculos/efeitos dos fármacos , Ratos , Frações Subcelulares/enzimologia , Fatores de Tempo
20.
Br J Cancer ; 83(8): 998-1002, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10993645

RESUMO

DT-diaphorase, a cytosolic reductase, has been implicated as an activator of chemotherapeutic prodrugs and a detoxifier of certain potentially carcinogenic xenobiotics. A common C to T nucleotide 609 substitution in DT-diaphorase cDNA has been associated with protein instability and reduced catalytic activity. The degree to which the allelic status of the substitution correlates with enzymatic activity was assessed in 45 normal human skin fibroblast strains using a PCR-RFLP assay. Included in this study was the 3437T strain, which is unique in that it is heterozygous for the polymorphism yet contains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP technique attributed this phenomenon to exclusive DT-diaphorase mRNA expression from the variant allele. Overlap in activities was observed between individual strains homozygous for the wild-type allele and heterozygotes, but the former group displayed enzymatic activity that was on average 2-fold higher. Western blot analysis of the two strains in this panel that are homozygous for the variant allele revealed that they express relatively low amounts of DT-diaphorase protein, consistent with the role of the substitution in protein instability. This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity.


Assuntos
NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Adolescente , Adulto , Linhagem Celular , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/enzimologia , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Pele/enzimologia
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