RESUMEN
DNA extracted from formalin-fixed and paraffin-embedded brain tissue is known to contain as yet ill-characterized inhibitors of the PCR process. As part of a project that aims to clarify the role of mitochondrial DNA sequence variation in human neurodegenerative diseases using DNA from various ethnic backgrounds, we have investigated factors that influence the preservation of archival DNA and its suitability for PCR. In this study, neuropathological tissue samples were analysed that had been routinely processed in 18 international centres on four continents. Following DNA extraction, PCR amplification of mitochondrial and nuclear DNA sequences was performed with and without additional purification of the template DNA. In addition, the DNA used for PCR was analysed by HPLC. Phosphate-buffered formalin proved to be a superior fixative compared with unbuffered aldehyde: DNA extraction resulted in greater yields, the molecular weight of the isolated DNA was higher and PCR was more successful. PCR inhibitors were identified as (1) high concentrations of small (<300 bp) DNA fragments that competitively compete with template DNA and (2) contaminants of the DNA template solution including denatured protein that cannot be completely removed by phenolic extraction. HPLC analysis did not reveal significant qualitative differences between DNA isolated from fresh-frozen tissue samples and DNA recovered from formalin-fixed, paraffin-embedded brain tissue. The fact that DNA could be amplified from the majority of tissue specimens in this study suggests that rare diseases and diseases where ethnic background plays an important role can be sampled for genetic polymorphism analysis on a global scale using archival neuropathological collections.
Asunto(s)
Química Encefálica , Encéfalo/patología , ADN Mitocondrial/aislamiento & purificación , ADN/aislamiento & purificación , Variación Genética , Laboratorios/normas , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Cromatografía Líquida de Alta Presión , ADN/genética , ADN Mitocondrial/genética , Síndrome de Down/genética , Síndrome de Down/patología , Humanos , Enfermedad por Cuerpos de Lewy/genética , Enfermedad por Cuerpos de Lewy/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Reacción en Cadena de la Polimerasa , Manejo de Especímenes/métodos , Conservación de Tejido/métodosRESUMEN
Cytosine methylation is an epigenetic modification of DNA involved in control of gene expression. Neoplastic cells exhibit various alterations both in DNA methylation and activity of the enzyme responsible for this modification, 5-methyltransferase (5-MeTase). As there is little requirement for 5-methyltransferase expression in normal cells except during mitosis, we argued that the gene would be hypermethylated in normal cells. Southern analysis revealed almost complete methylation of the gene in genomic DNA from the peripheral blood leukocytes of healthy subjects and a primary fibroblast derived cell line. In contrast, in DNA from a range of tumour tissues and tumour derived cell lines, 5-MeTase exhibited marked hypomethylation. The results of this study indicate that dysregulation of the DNA methylating machinery, especially with respect to the methylation status of 5-MeTase, is a feature of a wide range of neoplasms.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN de Neoplasias/metabolismo , Neoplasias/genética , Southern Blotting , Ciclo Celular/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Células Tumorales CultivadasRESUMEN
The venom of many dangerous Australian snakes has a myotoxic component and some are strongly myolytic. The myotoxicity of venom of seven Australian elapid snakes was studied to determine their relative in vitro potency in causing cell death of C2C12 cells, a myoblast cell line, and murine pmyotubes in mixed cell culture. The venom of Pseudechis australis proved to be most myotoxic, Austrelaps superbus and Pseudechis porphyriacus venoms also exhibited myotoxicity relative to the other venoms tested. The specificity of Pseudechis porphyriacus venom was tested using the human glioma cell line TC3 and was shown to exhibit a general cytotoxicity. Myotoxicity, however, was the predominant action of the venom. It has long been known that certain animals sucha as the mongoose (herpestes edwardsii) are able to survive envenomation. Some species of snakes also possess this property and the neutralising factor(s) responsible for this P. porphyriacus has been shown to be present in the serum. The protective effect of homologous plasma from P. porphyriacus venom was also studied with reference to myotoxicity and cytotoxicity. The results of this study clearly demonstrated protection by homologous plasma using a myoblast cell line, C2C12, a primary mixed cell culture and TC3 cells. While protection was clear, particularly using high concentrations of venom, it was not absolute, and homologous plasma did not afford continued protection from the effects of the venom. In the mixed culture experiments venom/plasma mixtures pre-incubated for 30 min were more protective than venom/plasma mixtures which were not incubated, in contrast to the results of cell culture studies, which showed little difference.
Asunto(s)
Humanos , Animales , Glioma , Técnicas In Vitro , Músculos/patología , Toxinas Biológicas/farmacología , Tifus Endémico Transmitido por Pulgas , Venenos Elapídicos/análisis , Antivenenos , AustraliaRESUMEN
Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Factores de Transcripción , Animales , Northern Blotting , Southern Blotting , Sondas de ADN , Enzimas de Restricción del ADN/metabolismo , Ratones , Proteínas Musculares/química , Proteínas del Tejido Nervioso/química , Factor de Transcripción PAX3 , Factor de Transcripción PAX7 , Factores de Transcripción Paired Box , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Previous studies have indicated that there may be uncleavable TaqI sites (TCGA) within the mouse myogenic gene, Myo-D1. Fragments of DNA bearing most of the presumed insensitive TaqI sites have been reproduced using PCR. The presence of each of the originally uncleavable TaqI sites has been confirmed and each TaqI site has been shown to be sensitive to TaqI hydrolysis in PCR-synthesized genomic DNA. Since TaqI is inhibited by methylation of the adenine residue within its recognition sequence (but not by cytosine methylation), it is suggested that specific adenine bases are methylated in the coding region of Myo-DI and maintained throughout cell division. The same TaqI recognition sequences are insensitive to digestion in genomic DNA isolated from various mouse tissues including fetus, regenerating skeletal muscle and a myogenic cell line, all of which express Myo-D1. Thus, adenine methylation is not a modification of DNA following gametic fusion nor does it appear to play a major role in regulation of Myo-D1 expression.
Asunto(s)
ADN/metabolismo , Ratones/genética , Proteína MioD/genética , Adenina , Animales , Secuencia de Bases , ADN/genética , Cartilla de ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal , Epidídimo , Femenino , Edad Gestacional , Hígado/metabolismo , Masculino , Metilación , Ratones Endogámicos A , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Músculos/metabolismo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Espermatozoides/metabolismo , Transactivadores/genéticaRESUMEN
A common technique used in the study of human malignancies involves the inoculation of nude mice with human neoplastic cells. It is usually assumed that the tumor arising is composed predominantly of human cells with mouse tissue present only to provide minimal stromal support. Several reports, however, have shown evidence of host cell neoplastic transformation. Therefore, in order to effectively study and characterize such xenografts, it is important to establish the relative involvement of human and mouse cells. In the present study, a method for easily distinguishing human and mouse cells is described. The method involves in situ hybridization of formalin-fixed tissues using DIG-labeled oligomer probes which correspond to species-specific portions of Alu sequences. This method can be applied to archival material either as a means of confirming that the tissue taken from nude mice xenografts is predominantly human or as a vehicle for studying the mechanisms of host cell neoplastic transformation and their relevance to human malignant spread. The proposed technique may also serve as a basis for other in situ applications, particularly those involving formalin-fixed tissues and oligomer probes.
Asunto(s)
Transmisión de Enfermedad Infecciosa , Hibridación in Situ/métodos , Trasplante de Neoplasias/patología , Animales , Secuencia de Bases , Transformación Celular Neoplásica , Humanos , Ratones , Datos de Secuencia Molecular , Adhesión en Parafina , Coloración y EtiquetadoRESUMEN
Binding of polyclonal antibodies specific for bFGF was examined in tissue sections of myopathic and normal muscles from humans, dogs and mice. The proposal tested was that differences in the amount or distribution of bFGF in muscles of the 3 species, might correlate with the limited muscle regeneration seen in humans and dogs afflicted with x-linked muscular dystrophy, in contrast with the sustained new muscle formation in mdx mice with the homologous myopathy. There was a striking difference between the species in the binding of bFGF antibodies to extracellular matrix, particularly at the periphery of myofibres; binding was pronounced in mouse but weak or absent in human and dog muscle. Binding to muscle nuclei and sarcoplasm was also stronger in mice than in humans and dogs, and in all species was more pronounced in foetal than adult muscle. Increased binding of bFGF antibodies was seen in damaged and regenerating muscle cells in all myopathic specimens where these were present. This was associated with the regenerative process rather than with myopathy, as a similar pattern of bFGF expression was seen in mouse muscle regenerating after experimental crush injury. The higher extracellular staining for bFGF around the periphery of mouse myofibres correlated with the successful muscle regeneration in dystrophic mice. Results suggest that bFGF at the fibre periphery might stimulate a local increase in the numbers of muscle precursor cells which can respond to injury in the mdx mouse.
Asunto(s)
Distrofina/deficiencia , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular Animal/metabolismo , Adolescente , Adulto , Animales , Niño , Preescolar , Perros , Femenino , Factor 2 de Crecimiento de Fibroblastos/inmunología , Ligamiento Genético , Humanos , Inmunohistoquímica , Lactante , Masculino , Ratones , Ratones Mutantes , Músculos/patología , Músculos/fisiología , Distrofias Musculares/genética , Distrofias Musculares/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Regeneración , Especificidad de la Especie , Cromosoma XRESUMEN
Genetic polymorphism of the epidermal growth factor (EGF) receptor gene following Taq I digestion was compared between samples of genomic DNA from glioma-derived cell lines and Caucasoid and Japanese subjects. The same three allelic forms of the EGF receptor gene, marked by variant fragments of approximately 12.8, 11.6 and 10.8 kb in size were common to both ethnic groups and the 12.8- and 11.6-kb fragments were found in the glioma-derived cell line DNA. A further variant fragment of approximately 13.8 kb in size has been shown to be thus far restricted to the Japanese. These data suggest that most allelic forms of the EGF receptor gene recognized by Taq I restriction fragment length polymorphism have a long evolutionary history and probably do not predispose to development of malignant glioma.
Asunto(s)
Receptores ErbB/genética , Polimorfismo Genético , Alelos , Pueblo Asiatico/genética , Southern Blotting , Frecuencia de los Genes , Genes , Humanos , Japón , Fenotipo , Población Blanca/genéticaRESUMEN
Polymorphism of the myogenic gene, Myo-D1, has been sought to examine genetic mechanisms which control skeletal muscle development. By Southern analysis, three restriction-fragment length polymorphisms (RFLPs) have been found in various mouse strains using the TaqI, SacI and BglII restriction endonucleases and a full-length cDNA Myo-D1 probe. Reference to the distribution of RFLPs in different mouse strains derived from Mus mus (M.m.) domesticus and M.m. musculus subspecies suggests that Myo-D1 rearrangements are subject to nonrandom association. The biological significance of RFLP of the Myo-D1 gene is yet to be determined.
Asunto(s)
Ratones Endogámicos/genética , Proteína MioD , Proteínas Nucleares/genética , Fosfoproteínas/genética , Polimorfismo Genético , Transactivadores/genética , Alelos , Animales , Southern Blotting , ADN/análisis , Reordenamiento Génico , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Polimorfismo de Longitud del Fragmento de RestricciónAsunto(s)
Proteínas Musculares/análisis , Distrofia Muscular Animal/metabolismo , Enfermedades de las Ovejas/metabolismo , Animales , Distrofina , Técnica del Anticuerpo Fluorescente , Secciones por Congelación , Inmunohistoquímica , Masculino , Proteínas Musculares/inmunología , Músculos/análisis , OvinosRESUMEN
Tumor formation in nude mice (nu/nu Balb c outbred) inoculated with cells from four new permanent human glioma cell lines was studied. Three of these lines had previously been shown to display features of striated muscle in vitro. Histochemical and immunochemical techniques together with electron microscopic study confirmed that striated muscle differentiation continued to be expressed in vivo. Two of the cell lines arguably showed greater striated muscle differentiation in vivo, whereas one has lost this ability. In one of the two, further mesodermal differentiation was evident with the formation of cartilage.
Asunto(s)
Glioma/patología , Animales , Diferenciación Celular , Línea Celular , Citoplasma/patología , Glioma/análisis , Glioma/ultraestructura , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Músculos/patología , Trasplante de NeoplasiasRESUMEN
Neurophysiological studies suggested that transynaptic neuronal degeneration of the anterior horn cells (AHC) may occur after an upper motoneuron lesion as the result of "deafferentation". To test this observation anatomically, patients with spinal cord injury (SCI) who had come to post mortem were investigated. Four patients with longstanding clinically and pathologically "complete" SCI were selected for comparison with 4 age-matched normal controls and with 2 patients who died of motoneuron disease (MND). The total number of AHCs in the L3 spinal cord segment was counted in each of the cases. The lesions in the traumatic group were all above the L3 segment. No significant differences in the number of AHC between the test cases and the normal controls was found. There was, as expected, a highly significant difference between the test cases and those with MND. The conclusion drawn from the study is that transynaptic neuronal degeneration of AHCs does not occur following complete transection of the human spinal cord. Thus the neurophysiological hypothesis is not supported anatomically.
Asunto(s)
Neuronas Motoras/citología , Degeneración Nerviosa , Traumatismos de la Médula Espinal/patología , Sinapsis/fisiología , Humanos , Enfermedades Neuromusculares/patología , Paraplejía/patologíaRESUMEN
A series of bifunctional alkylators were tested against the genotypically and phenotypically heterogeneous continuous human medulloblastoma cell lines, TE-671, Daoy, and D283 Med in vitro and against TE-671 and Daoy growing as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (in vivo), and busulfan (in vivo). Melphalan and phenylketocyclophosphamide were the most active agents in vitro with drug doses at which there is a 90% reduction in the number of colonies in comparison to controls of 2.13, 5.29, and 4.72 microM for melphalan and 4.60, 5.01, and 4.34 microM for phenylketocyclophosphamide against TE-671, D283 Med, and Daoy, respectively. Melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, and thiotepa produced significant growth delays against s.c. TE-671 and Daoy xenografts, while no activity could be demonstrated for 1,3-bis(2-chloroethyl)-1-nitrosourea or busulfan. Melphalan, cyclophosphamide, iphosphamide, and thiotepa also produced significant increases in median survival in mice bearing intracranial TE-671 and Daoy xenografts. These results extend our previous studies demonstrating the antitumor activity of nitrogen and phosphoramide mustard-based bifunctional alkylating agents in the treatment of human medulloblastoma continuous cell lines and transplantable xenografts, and support the continued use of these agents in clinical trials.
Asunto(s)
Alquilantes/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Meduloblastoma/tratamiento farmacológico , Animales , Línea Celular , Reactivos de Enlaces Cruzados , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de NeoplasiasRESUMEN
A glioblastoma multiforme from a 64-year-old man was heterotransplanted sc into nude mice (nu/nu BALB/c out-bred). The subsequent growth of the transplanted tumor featured the appearance and eventual domination by cells with ependymal characteristics. This finding supports the view that the host's influence on heterotransplants may be substantial and should be considered when extrapolating the results of therapeutic trials on such animal models to the clinical situation. The view that ependymal cells and astrocytes share a common precursor is also partly supported by the observations.
Asunto(s)
Epéndimo/patología , Glioblastoma/patología , Animales , Diferenciación Celular , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
Twenty-two human gliomas were set up in tissue culture and inoculated into nude mice. Permanent cell lines were established from four of these and their growth characteristics, and morphological features defined and cytogenetic features described. All four failed to sustain evidence of glial differentiation while three of the four cell lines exhibited the characteristics of striated muscle, and were tumorigenic in nude mice. Nine gliomas showed some initial growth in nude mice but only two were successfully subpassaged.
Asunto(s)
Neoplasias Encefálicas/patología , Línea Celular/citología , Glioma/patología , Músculos/citología , Animales , Neoplasias Encefálicas/ultraestructura , Diferenciación Celular , Línea Celular/ultraestructura , Glioma/ultraestructura , Histocitoquímica , Humanos , Técnicas para Inmunoenzimas , Cariotipificación , Ratones , Ratones Desnudos , Músculos/ultraestructura , Proteínas de Neoplasias/análisis , Trasplante de NeoplasiasRESUMEN
A permanent cell line arising from a cerebellar medulloblastoma was established and its growth characteristics were investigated. Although the original tumor inoculum failed to take, the cultured cells were readily tumorigenic in nude mice and gave rise to rapidly growing tumors which could be easily subpassaged. The primary tumor showed evidence of both glial and neuronal differentiation, and retention of neuronal differentiation, albeit minimal, occurred in both the cultured neoplastic cells and the nude mouse tumors. Glial differentiation, on the other hand, could not be demonstrated. G-banding analysis of the chromosomes present in the cell line showed that they were exclusively human.
Asunto(s)
Neoplasias Cerebelosas/patología , Meduloblastoma/patología , Agar , Animales , Bucladesina/farmacología , Recuento de Células , División Celular , Línea Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/ultraestructura , Preescolar , Bandeo Cromosómico , Medios de Cultivo , Humanos , Cariotipificación , Masculino , Meduloblastoma/genética , Meduloblastoma/ultraestructura , Ratones , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
Clinicopathological data have been collected for 42 patients with cerebellar medulloblastoma diagnosed and treated in Western Australia between the years 1961 and 1984. Thirty-one patients were male and 12 were over 14 years of age. In 31 patients the neoplasms were localized to the midline, while in 10 they were placed laterally in the cerebellar hemispheres. The remaining patient had a diffusely spreading neoplasm in the meninges of the posterior fossa. Thirty-nine underwent surgical treatment followed by postoperative radiotherapy. Three patients died in the early postoperative period. Twenty-four patients survived for one year or longer, 15 for two or more years, 10 for five years, and five survived longer than 10 years. The survival proportions estimated by the life-table method were 66% at one year, 48% at two years and 32% at 5 and 10 years. In one patient who survived for 23 years a meningioma developed, possibly due to radiotherapy.
Asunto(s)
Neoplasias Cerebelosas/patología , Meduloblastoma/patología , Adolescente , Adulto , Factores de Edad , Neoplasias Cerebelosas/mortalidad , Neoplasias Cerebelosas/terapia , Cerebelo/patología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Meduloblastoma/mortalidad , Meduloblastoma/terapia , Meninges/patología , Factores de TiempoAsunto(s)
Músculos/patología , Distrofias Musculares/patología , Enfermedades del Sistema Nervioso Periférico/patología , Adulto , Anciano , Biopsia , Niño , Estudios de Evaluación como Asunto , Femenino , Tamización de Portadores Genéticos , Técnicas Histológicas , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Distrofias Musculares/genéticaRESUMEN
A cerebral neuroblastoma was studied by light microscopy, tissue culture and electron microscopy. Synapse-like structures were found within tumour cells. This supports the neuroectodermal origin of this neoplasm.