Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 30
Filter
Add more filters










Publication year range
1.
Radiother Oncol ; 61(3): 309-12, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11731001

ABSTRACT

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.


Subject(s)
Adenoviridae , Genes, p53 , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Nasopharyngeal Neoplasms/therapy , Adenoviridae/genetics , Animals , Genetic Vectors/genetics , Humans , Mice , Mice, Knockout , Nasopharyngeal Neoplasms/radiotherapy , Transplantation, Heterologous
2.
Hum Mol Genet ; 10(23): 2627-35, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11726549

ABSTRACT

X-linked dilated cardiomyopathy (XLDC) is a dystrophinopathy characterized by severe cardiomyopathy with no skeletal muscle involvement. Several XLDC patients have been described with mutations that abolish dystrophin muscle (M) isoform expression. The absence of skeletal muscle degeneration normally associated with loss of dystrophin function was shown to be due to increased expression of brain (B) and cerebellar Purkinje (CP) isoforms of the gene exclusively in the skeletal muscle of these patients. This suggested that the B and CP promoters have an inherent capacity to function in skeletal muscle or that they are up-regulated by a skeletal muscle-specific enhancer unaffected by the mutations in these patients. In this work we have analyzed the deletion breakpoints of two XLDC patients with deletions removing the M promoter and exon 1, but not affecting the B and CP promoters. Despite the presence of several muscle-specific regulatory motifs, the B and CP promoters were found to be essentially inactive in muscle cell lines and primary cultures. As dystrophin muscle enhancer 1 (DME1), the only known muscle-specific enhancer within the dystrophin gene, is preserved in these patients, we tested its ability to up-regulate the B and CP promoters in muscle cells. B and CP promoter activity was significantly increased in the presence of DME1, and more importantly, activation was observed exclusively in cells presenting a skeletal muscle phenotype. These results point to a role for DME1 in the induction of B and CP isoform expression in the skeletal muscle of XLDC patients defective for M isoform expression.


Subject(s)
Cardiomyopathy, Dilated/genetics , Dystrophin/genetics , Enhancer Elements, Genetic/genetics , X Chromosome/genetics , 3T3 Cells , Animals , Base Sequence , Brain/metabolism , Cell Line , Cerebellum/metabolism , Cloning, Molecular , DNA/chemistry , DNA/genetics , Exons/genetics , Gene Deletion , Gene Expression Regulation , Genetic Linkage , Humans , Mice , Molecular Sequence Data , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Purkinje Cells/metabolism , Sequence Analysis, DNA
3.
Br J Cancer ; 83(8): 998-1002, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10993645

ABSTRACT

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.


Subject(s)
NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , Point Mutation , Polymorphism, Restriction Fragment Length , Adolescent , Adult , Cell Line , Cells, Cultured , Child , Child, Preschool , Female , Fibroblasts/enzymology , Genotype , Humans , Infant , Infant, Newborn , Male , Polymerase Chain Reaction/methods , Polymorphism, Genetic , Skin/enzymology
4.
J Biol Chem ; 275(26): 20127-35, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10777512

ABSTRACT

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.


Subject(s)
Apoptosis , DNA Damage , Growth Substances/metabolism , Pregnancy Proteins/metabolism , Tumor Suppressor Protein p53/biosynthesis , Adenoviridae/metabolism , Blotting, Northern , Breast Neoplasms/metabolism , Cell Cycle/drug effects , Cell Division , Cell Survival/drug effects , DNA, Complementary/metabolism , Genes, p53/genetics , Genetic Vectors/metabolism , Humans , RNA, Messenger/metabolism , Sequence Analysis, DNA , Time Factors , Tissue Distribution , Transcriptional Activation , Tumor Cells, Cultured , Up-Regulation/drug effects , Up-Regulation/radiation effects
5.
Neuromuscul Disord ; 10(3): 187-93, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10734266

ABSTRACT

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.


Subject(s)
Alternative Splicing/genetics , Brain Chemistry/genetics , Dystrophin/analogs & derivatives , Muscular Dystrophy, Duchenne/genetics , Cloning, Molecular , Dystrophin/genetics , Gene Expression Regulation , Humans , Immunoblotting , Polymerase Chain Reaction , RNA, Messenger/genetics , Transcription, Genetic
6.
Cancer Gene Ther ; 7(12): 1566-74, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11228535

ABSTRACT

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.


Subject(s)
Adenoviridae/genetics , Gene Targeting/methods , Genetic Therapy/methods , Genetic Vectors , Hyperthermia, Induced , Tumor Cells, Cultured/metabolism , Animals , Female , Gene Expression , Genes, Reporter , HSP70 Heat-Shock Proteins/genetics , Humans , Interleukin-12/genetics , Mice , Mice, SCID , Transfection , beta-Galactosidase/metabolism
7.
Mutat Res ; 406(2-4): 45-54, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10479721

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Monomeric GTP-Binding Proteins , Nucleoside-Diphosphate Kinase , Analysis of Variance , Animals , Cell Extracts/genetics , Cell Line , DNA Probes , DNA, Complementary/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , NM23 Nucleoside Diphosphate Kinases , RNA, Messenger/genetics , Receptors, Cell Surface/genetics , Receptors, Urokinase Plasminogen Activator , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Ribosomal Proteins/genetics , Transcription Factors/genetics , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/metabolism
8.
Cancer Gene Ther ; 6(2): 179-90, 1999.
Article in English | MEDLINE | ID: mdl-10195885

ABSTRACT

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.


Subject(s)
Breast Neoplasms/therapy , Ganciclovir/therapeutic use , Genetic Therapy , Simplexvirus/genetics , Thymidine Kinase/genetics , Acridine Orange/metabolism , Adenoviridae/metabolism , Apoptosis , Blotting, Northern , Blotting, Western , Cell Survival , DNA Fragmentation , Ethidium/metabolism , Fluorescent Dyes/metabolism , Humans , Proto-Oncogene Proteins c-bcl-2/metabolism , Sensitivity and Specificity , Thymidine Kinase/administration & dosage , Time Factors , Transduction, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/metabolism
9.
Muscle Nerve ; 22(1): 16-27, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9883853

ABSTRACT

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells. The localization of Dp71 in these cells is distinct from the localization of Dp427. Whereas Dp427 localizes to focal adhesions and surface membrane during myogenesis, Dp71 localizes to stress fiberlike structures in myogenic cells. Biochemical fractionation of myogenic cells demonstrates that Dp71 cosediments with the actin bundles thus confirming this interaction. Furthermore, transfection of C2C12 myoblasts with constructs encoding Dp71 fused to green fluorescent protein targeted the protein to the actin microfilament bundles. These results demonstrate involvement of Dp71 with the actin cytoskeleton during myogenesis and suggest a role for Dp71 that is distinct from Dp427.


Subject(s)
Dystrophin/analogs & derivatives , Dystrophin/physiology , Muscle, Skeletal/metabolism , Actins/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Dystrophin/analysis , Dystrophin/genetics , Fetus , Gene Expression Regulation, Developmental , Green Fluorescent Proteins , Humans , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Microscopy, Fluorescence , Muscle, Skeletal/chemistry , Protein Isoforms , Recombinant Fusion Proteins , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions , Transfection
10.
Int J Radiat Oncol Biol Phys ; 42(2): 331-4, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9788412

ABSTRACT

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.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Anus Neoplasms/drug therapy , Anus Neoplasms/radiotherapy , NAD(P)H Dehydrogenase (Quinone)/genetics , Neoplasm Proteins/genetics , Anus Neoplasms/enzymology , Anus Neoplasms/genetics , Combined Modality Therapy , Fluorouracil/administration & dosage , Humans , Mitomycin/administration & dosage , Polymorphism, Genetic , Retrospective Studies , Treatment Outcome
11.
Breast Cancer Res Treat ; 48(3): 273-86, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9598874

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Genetic Therapy , Tumor Suppressor Protein p53/therapeutic use , Adenoviridae/genetics , Breast Neoplasms/therapy , Female , Gene Transfer Techniques , Humans , Tumor Cells, Cultured , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics
12.
Br J Cancer ; 77(8): 1236-40, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9579828

ABSTRACT

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.


Subject(s)
COS Cells/enzymology , DNA, Complementary/genetics , Gene Expression Regulation, Enzymologic/genetics , NAD(P)H Dehydrogenase (Quinone)/genetics , Point Mutation , Transfection , Animals , Base Composition , Blotting, Western , Cells, Cultured , Chlorocebus aethiops , DNA Primers/chemistry , Humans , NAD(P)H Dehydrogenase (Quinone)/metabolism , Polymorphism, Genetic
13.
FEBS Lett ; 441(2): 337-41, 1998 Dec 18.
Article in English | MEDLINE | ID: mdl-9883911

ABSTRACT

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.


Subject(s)
Actins/metabolism , Dystrophin/analogs & derivatives , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Cytoskeleton/metabolism , Dystrophin/chemistry , Dystrophin/metabolism , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
14.
Gene ; 200(1-2): 173-6, 1997 Oct 24.
Article in English | MEDLINE | ID: mdl-9373152

ABSTRACT

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.


Subject(s)
Brain/metabolism , Dystrophin/genetics , Evolution, Molecular , Muscle, Skeletal/metabolism , Promoter Regions, Genetic , Transcription Factor AP-1/metabolism , Transcription, Genetic , Base Sequence , Binding Sites , Brain/embryology , Cerebellum/metabolism , Conserved Sequence , Exons , Fetus , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Purkinje Cells/metabolism , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Sequence Tagged Sites
15.
Nucleic Acids Res ; 25(8): 1618-25, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9092671

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/metabolism , Dystrophin/biosynthesis , Dystrophin/genetics , Enhancer Elements, Genetic , Introns , Muscle, Skeletal/metabolism , MyoD Protein/metabolism , Regulatory Sequences, Nucleic Acid , Transcription Factors/metabolism , Adenine , Base Composition , Base Sequence , Binding Sites , Brain/metabolism , Cell Line , Cerebellum/metabolism , DNA Primers , Genes, Reporter , Humans , MEF2 Transcription Factors , Molecular Sequence Data , Myogenic Regulatory Factors , Polymerase Chain Reaction , Purkinje Cells/metabolism , RNA, Messenger/biosynthesis , Recombinant Proteins/biosynthesis , Restriction Mapping , Thymine , Transcription, Genetic
16.
Genomics ; 40(2): 294-304, 1997 Mar 01.
Article in English | MEDLINE | ID: mdl-9119397

ABSTRACT

The sequence of a 112-kb region of the human dystrophin (DMD/BMD) gene encompassing the deletion prone intron 7 (110 kb) and the much shorter intron 8 (1.1 kb) has been determined. Recognizable insertion sequences account for approximately 40% of intron 7. LINE-1 and THE-1/LTR sequences occur in intron 7 with significantly higher frequency than would be expected statistically while Alu sequences are underrepresented. Intron 7 also contains numerous mammalian-wide interspersed repeats, a diverse range of medium reiteration repeats of unknown origin, and a sequence derived from a mariner transposon. By contrast, the shorter intron 8 contains no detectable insertion sequences. Dating of the LI and Alu sequences suggests that intron 7 has approximately doubled in size within the past 130 million years, and comparison with the corresponding intron from the pufferfish (Fugu rubripes) suggests that the intron has expanded some 44-fold over a period of 400 million years. The possible contribution of the insertion elements to the instability of intron 7 is discussed.


Subject(s)
Dystrophin/genetics , Evolution, Molecular , Introns/genetics , Base Composition , Genes/genetics , Humans , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid/genetics , Sequence Analysis, DNA , Sequence Deletion/genetics
17.
Hum Mol Genet ; 5(10): 1599-606, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8894694

ABSTRACT

The 14 kb muscle isoform of the Duchenne muscular dystrophy (DMD) gene is expressed primarily in skeletal and cardiac muscle. Transcription of the muscle isoform is induced as myoblasts differentiate into multinucleated myotubes and transcript levels are increased a further 10-fold in mature skeletal muscle. In previous studies we have demonstrated that the core muscle promoter of the human DMD gene contains sequences that regulate the induction of DMD gene expression with myoblast differentiation. However, direct injection studies have indicated that the activity of the core muscle promoter in mature skeletal muscle is 30-fold lower than in immature myotubes. This discrepancy between endogenous transcript levels and core promoter activity suggested that additional transcriptional elements are involved in the regulation of DMD gene expression in muscle. In this report we present evidence for the existence of a muscle-specific enhancer within intron 1 of the human DMD gene. Functional analysis of Hindill fragments from within a 36 kb region surrounding muscle exon 1 of the human DMD gene resulted in the identification of a 5.0 kb fragment within muscle intron 1 that consistently provided high levels of reporter gene expression in both immature and mature skeletal muscle. Sequences within this 5 kb fragment were shown to be functionally independent of position and orientation and to be inactive in fibroblasts, properties that are consistent with the definition of a muscle-specific enhancer. Although this enhancer provided a 30-fold increase in transcription from a SV40 viral promoter in mature skeletal muscle, only a 3-fold increase was observed from the DMD core muscle promoter. Intron 1 enhancer activity alone is therefore insufficient to account for the discrepancy between endogenous transcript levels and core muscle promoter activity in immature and mature skeletal muscle and points to the existence of additional enhancer elements in other regions of the DMD gene. This report provides the first evidence for the involvement of a transcriptional enhancer in DMD gene regulation in muscle and impacts on our understanding of the functional consequences of mutations at the 5'-end of gene. In this regard, deletions in this region in X-linked dilated cardiomyopathy patients provides indirect evidence for a role for this enhancer in regulating DMD gene expression in cardiac muscle.


Subject(s)
Dystrophin/genetics , Enhancer Elements, Genetic/genetics , Genome, Human , Introns/genetics , Muscular Dystrophies/genetics , Humans , Muscle, Skeletal/metabolism , Muscular Dystrophies/metabolism , Sequence Analysis , Transcription, Genetic
18.
Hum Mol Genet ; 4(9): 1475-83, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8541829

ABSTRACT

Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues. In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes. The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78. RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver. To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins. These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B). The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values. Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues. This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78. Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain beta-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC).(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Alternative Splicing , Dystrophin/analogs & derivatives , Adult , Amino Acid Sequence , Amnion/cytology , Amnion/metabolism , Base Sequence , Cells, Cultured , Cloning, Molecular , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA, Complementary , Dystroglycans , Dystrophin/genetics , Dystrophin/metabolism , Escherichia coli/genetics , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism
19.
Nat Genet ; 9(2): 184-90, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7719347

ABSTRACT

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.


Subject(s)
Dystrophin/genetics , Transcription, Genetic , Animals , Base Sequence , Cell Differentiation , DNA, Recombinant , Exons , Gene Expression , Humans , Molecular Sequence Data , Muscles/cytology , Polymerase Chain Reaction/methods , Time Factors
20.
Exp Cell Res ; 192(1): 278-88, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1984418

ABSTRACT

Sarcolemmal vesicles with right-side-out configuration were prepared from normal fresh human and rabbit skeletal muscle bundles by incubation in 140 mM KCl solution containing collagenase. The vesicles were used to examine the association of dystrophin, the protein product of the Duchenne muscular dystrophy gene, with the sarcolemma. Western blot analysis, indirect immunofluorescence, and immunoperoxidase staining using specific antibodies raised against the N-terminal and the C-terminal domains show that dystrophin remains associated with the membrane of sarcolemmal vesicles. Indirect immunofluorescence microscopy using permeabilized and unpermeabilized vesicles indicated that both the N-terminus and the C-terminus of dystrophin are localized to the cytoplasmic surface of the sarcolemma. These results suggest that dystrophin has much stronger attachment to the surface membrane than it has to the internal domain of skeletal muscle fibers. Sarcolemmal vesicles thus represent a new system for studying the function of dystrophin and the molecular basis of its association with the sarcolemma.


Subject(s)
Dystrophin/analysis , Sarcolemma/chemistry , Amino Acid Sequence , Animals , Cell Fractionation/methods , Cytoplasm/chemistry , Immunoblotting , Immunohistochemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...