RESUMO
Based on previous observations that capsaicin can selectively damage group III and IV afferents and induce muscle fibre transformation, we hypothesized that eliminating, by means of capsaicin, the group III and IV afferents of a peripheral territory it could lead to a fibre transformation in a muscle involved in the flexor reflexes of the same peripheral territory. Therefore, capsaicin was injected into the palmar nerves of the forelimb of the horse to investigate if eliminating group III and IV afferents from the hand of the horse a muscle fibre transition would occur in the flexor carpi radialis (FCR) muscle, which is involved in the flexor reflexes of the finger itself. 120 days after capsaicin injection, type I slow fibres increased and type IIA fast fibres decreased. We presume that the long lasting deafferentation of the ergo-nociceptive fibres causes a plastic remodelling in the central nervous system and indirectly influences the motoneuron excitability via short or long loop-pathways enhancing their tonic discharge.
Assuntos
Membro Anterior/inervação , Denervação Muscular , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Pele/inervação , Animais , Capsaicina/farmacologia , Cavalos , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/ultraestrutura , Fibras Musculares de Contração Lenta/enzimologia , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/efeitos dos fármacosRESUMO
The role of muscle ischemia and fatigue in modulating the monosynaptic reflex was investigated in decerebrate and spinalized rats. Field potentials and fast motoneuron single units in the lateral gastrocnemious (LG) motor pool were evoked by dorsal root stimulation. Muscle ischemia was induced by occluding the LG vascular supply and muscle fatigue by prolonged tetanic electrical stimulation of the LG motor nerve. Under muscle ischemia the monosynaptic reflex was facilitated since the size of the early and late waves of the field potential and the excitability of the motoneuron units increased. This effect was abolished after L3-L6 dorsal rhizotomy, but it was unaffected after L3-L6 ventral rhizotomy. By contrast, the monosynaptic reflex was inhibited by muscle fatiguing stimulation, and this effect did not fully depend on the integrity of the dorsal root. However, when ischemia was combined with repetitive tetanic muscle stimulation the inhibitory effect of fatigue was significantly enhanced. Both the ischemia and fatigue effects were abolished by capsaicin injected into the LG muscle at a dose that blocked a large number of group III and IV muscle afferents. We concluded that muscle ischemia and fatigue activate different groups of muscle afferents that are both sensitive to capsaicin, but enter the spinal cord through different roots. They are responsible for opposite effects, when given separately: facilitation during ischemia and inhibition during fatigue; however, in combination, ischemia enhances the responsiveness of the afferent fibres to fatigue.
Assuntos
Capsaicina/farmacologia , Isquemia/metabolismo , Neurônios Motores/efeitos dos fármacos , Fadiga Muscular/fisiologia , Músculo Esquelético/inervação , Reflexo Monosináptico/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Eletrofisiologia , Masculino , Potenciais da Membrana/fisiologia , Neurônios Motores/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Reflexo Monosináptico/fisiologiaRESUMO
Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Mutação em Linhagem Germinativa , Melanoma/genética , Proteínas Proto-Oncogênicas , Neoplasias Cutâneas/genética , Adulto , Idoso , Ciclo Celular , Divisão Celular , Quinase 4 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Análise Mutacional de DNA , DNA de Neoplasias/análise , Suscetibilidade a Doenças , Éxons , Feminino , Fase G1 , Ligação Genética , Testes Genéticos , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Transcrição GênicaRESUMO
MDM2 gene is overexpressed in several tumors and its product may be processed into different isoforms, some of which have been demonstrated to possess transforming activity. In a panel of liposarcomas characterized by displaying 4 different combinations of mdm2/p53 immunoreactivity, molecular analysis of amplified MDM2 gene revealed a coexistence of mutated full-length MDM2 messenger RNAs, an out-of-frame splicing mRNA and finally aberrant spliced forms. Two of the latter are reported here for the first time. The molecular differences in this heterogeneous mRNA population seem to mirror distinct functional aspects of the altered encoded mdm2 proteins. In fact, besides the deleted transcripts defective in their ability to bind p53 and known to possess a transforming activity, here we describe both mutated full-length forms and deleted transcripts that still maintain the ability to bind p53 but, based on their mdm2+/p53+ immunophenotype, probably fail to signal its degradation. These aberrant forms, which are responsible for the accumulation and inactivation of p53, can contribute, together with the p53 independent transforming forms, to liposarcoma transforming pathway.
Assuntos
Genes p53/genética , Lipossarcoma/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Abdominais/genética , Neoplasias Abdominais/metabolismo , Tecido Adiposo/metabolismo , Processamento Alternativo , Southern Blotting , Clonagem Molecular , Colo/metabolismo , Deleção de Genes , Humanos , Imunofenotipagem , Modelos Genéticos , Mutação , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Mutação Puntual , Reação em Cadeia da Polimerase , Testes de Precipitina , Biossíntese de Proteínas , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Torácicas/genética , Neoplasias Torácicas/metabolismoRESUMO
Supernumerary rings in the context of a simple karyotype characterize several low-grade malignant tumors of soft tissue and bone. Low-grade fibromyxoid sarcoma is an uncommon low-grade sarcoma, the cytogenetics of which has not yet been reported. Here we describe the first molecular-cytogenetic characterization of a pulmonary metastasis of low-grade fibromyxoid sarcoma. The histology of the primary and recurrent tumors was consistent with the diagnosis of low-grade fibromyxoid sarcoma of the usual type, whereas the pulmonary metastasis was of the "giant rosettes" variant. Cytogenetic analysis revealed a ring chromosome. Because gain of material of chromosomes 7 and 16 was detected by CGH, the ring chromosome is assumed to be composed of material from these respective chromosomes.
Assuntos
Fibrossarcoma/genética , Cromossomos em Anel , Neoplasias de Tecidos Moles/genética , Adulto , Southern Blotting , Aberrações Cromossômicas , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 7/genética , DNA de Neoplasias/genética , Feminino , Fibrossarcoma/patologia , Humanos , Cariotipagem , Hibridização de Ácido Nucleico , Neoplasias de Tecidos Moles/patologiaRESUMO
Tumor suppressor protein p53 is a positive regulator of MDM2 gene expression and the mdm2 protein can bind to p53, preventing the transactivation of p53 responsive genes, thus mimicking TP53 mutation. The authors looked for alterations that could affect, directly and indirectly, p53 function in 13 patients with extrahepatic cholangiocarcinoma. Molecular analysis by single strand conformation polymorphism and DNA sequencing revealed that TP53 gene mutations occurred in only 2 of 13 cholangiocarcinomas. High levels of mdm2 protein were found, by immunohistochemical staining, in 61% of the cholangiocarcinomas and in almost all specimens (70%) displaying stabilized p53 protein in the absence and in the presence of TP53 mutations. The finding of co-overexpressed mdm2 and p53 proteins in cholangiocarcinomas indicates that they can upregulate the expression of mdm2 protein to a level sufficient for binding and accumulating p53 in a presumably inactive complexed form. The presence of TP53 mutations or upregulation of MDM2 gene expression in 9 of the 13 cholangiocarcinomas strongly supports that the impairment of the p53 pathway is an important and specific step in cholangiocarcinoma pathogenesis. At variance with other authors, no alteration of p16ink4/CDKN2 gene was observed in all 13 cholangiocarcinomas.
Assuntos
Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/genética , Mutação , Proteínas Nucleares , Proteínas Proto-Oncogênicas/biossíntese , Proteína Supressora de Tumor p53/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA de Neoplasias/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas c-mdm2 , Análise de Sequência de DNARESUMO
Ordinary lipomas are cytogenetically characterized by a variety of balanced rearrangements involving chromosome segment 12q13-15, whereas well differentiated liposarcomas (WDL) show supernumerary ring and giant marker chromosomes, known to contain amplified 12q sequences. The tight correlation between the presence of ring chromosomes and both amplification and overexpression of MDM2 and CDK4 genes suggests the exploration of the possibility that immunocytochemistry (ICC) might assist in the differential diagnosis of lipoma-like well differentiated liposarcomas (LL-WDL) and large deep-seated lipomas (LDSL). For this purpose, 21 cases of the former and 19 cases of the latter tumours were analysed by ICC and, according to the availability of material, by molecular and cytogenetic approaches. All lipomas displayed a null MDM2/CDK4 phenotype, whereas all LL-WDL showed MDM2/CDK4 or CDK4 phenotypes. Southern blot analysis performed on 16 suitable cases, complemented by fluorescence in situ hybridization and classical cytogenetic analysis in 11 cases, was consistent with, and further supported the immunophenotyping data. In conclusion, MDM2/CDK4 product-based immunophenotyping appears to represent a valuable method for the categorization of arguable LDSL.
Assuntos
Lipoma/diagnóstico , Lipoma/genética , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , Quinases Ciclina-Dependentes/genética , Diagnóstico Diferencial , Feminino , Amplificação de Genes , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-IdadeRESUMO
Recent observations indicate the existence of pathogenetically distinct groups of well-differentiated (WD) dedifferentiated (DD) liposarcomas. In the retroperitoneal WD-DD liposarcomas, the predominant phenotype is represented by the aberrant (overexpressed) mdm2+/p53+ wild-type profile. At the nonretroperitoneal site, the WD liposarcomas present a wider association of MDM2/P53 gene expression; i.e., mdm2+/p53+, mdm2+/p53-, mdm2-/p53+ and mdm2-/p53-, and TP53 mutations seem to correlate with the dedifferentiation process. A biochemical study of mdm2-p53 association in 11 tumor samples characterized by the presence of different mdm2 and p53 immunophenotypes was performed. Immunoprecipitation assays using a p53-specific antibody were performed on tumor tissue and surrounding normal tissue; the immunoprecipitated material was then investigated for the presence of p53 (control) and of coimmunoprecipitated mdm2. This biochemical analysis showed that, in mdm2+/p53+/wild-type retroperitoneal liposarcomas, a band corresponded to mdm2 protein in the cellular lysates immunoprecipitated with a p53-directed antibody. In contrast, the mdm2+/p53- liposarcoma did not evidence the presence of mdm2 protein nor was p53 protein available to direct immunoprecipitation, as in the p53 mutant tumor samples with mdm2-/p53+ and mdm2-/p53- phenotypes. From the normal counterpart of retroperitoneal liposarcoma lysates, no p53 protein was immunoprecipitated. The findings in this study agree with the molecular data and they show the physical association of mdm2 and p53 in fresh liposarcoma surgical specimens.
Assuntos
Lipossarcoma/genética , Lipossarcoma/patologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Amplificação de Genes , Genes p53 , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-mdm2 , Neoplasias Retroperitoneais/genética , Neoplasias Retroperitoneais/patologia , Proteína Supressora de Tumor p53/análiseRESUMO
1. The role of group III and IV afferent fibres of the lateral gastrocnemious muscle (LG) in modulating the homonymous monosynaptic reflex was investigated during muscle fatigue in spinalized rats. 2. Muscle fatigue was induced by a series of increasing tetanic electrical stimuli (85 Hz, 600 ms) delivered to the LG muscle nerve. Series consisted of increasing train numbers from 1 to 60. 3. Potentials from the spinal cord LG motor pool and from the ventral root were recorded in response to proprioceptive afferent stimulation and analysed before and during tetanic muscle activations. Both the pre- and postsynaptic waves showed an initial enhancement and, after a '12-train' series, an increasing inhibition. 4. The enhancement of the responses to muscle fatiguing stimulation disappeared after L3-L6 dorsal root section, while a partial reflex inhibition was still present. Conversely, after section of the corresponding ventral root, there was only a reduction in the inhibitory effect. 5. The monosynaptic reflex was also studied in animals in which a large number of group III and IV muscle afferents were eliminated by injecting capsaicin (10 mM) into the LG muscle. As a result of capsaicin treatment, the fatigue-induced inhibition of the pre- and postsynaptic waves disappeared, while the response enhancement remained. 6. We concluded that the monosynaptic reflex inhibition, but not the enhancement, was mediated by those group III and IV muscle afferents that are sensitive to the toxic action of capsaicin. The afferents that are responsible for the response enhancement enter the spinal cord through the dorsal root, while those responsible for the inhibition enter the spinal cord through both the ventral and dorsal roots.
Assuntos
Capsaicina/farmacologia , Fadiga/fisiopatologia , Músculo Esquelético/inervação , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Reflexo Monosináptico/fisiologia , Animais , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Injeções Intramusculares , Masculino , Contração Muscular/fisiologia , Músculo Esquelético/fisiopatologia , Ratos , Ratos Wistar , Rizotomia , Medula Espinal/fisiopatologia , Raízes Nervosas Espinhais/fisiologiaRESUMO
It has recently been shown that mdm2 overexpression with stabilization of p53 represents a characteristic of retroperitoneal well-differentiated-dedifferentiated, here renamed evolved (WD-E), liposarcomas at the immunocytochemical, molecular, and cytogenetic level. This make-up appears to be confined to half the cases in non-retroperitoneal well-differentiated liposarcomas. Since in different tumours MDM2 amplification involves amplicons encompassing flanking genes, such as CDK4, the possibility was investigated that in these tumours, CDK4 could act as an alternative or additional gene involved in the transformation mechanism. Forty-one retroperitoneal (R)/non-retroperitoneal (NR) well-differentiated-dedifferentiated (WD-DD) and 33 myxoid/round cell liposarcomas were reanalysed by immunocytochemical, molecular (nine cases) and fluorescence in situ hybridization (FISH) (one case) techniques. The results showed that all but one R WD-E cases carried the mdm2+, p53+, cdk4+ immunophenotype. In NR-WD liposarcomas, this immunophenotype was shared in five cases and the remainder showed mdm2+, p53-, cdk4+ in four and mdm2-, p53-, cdk4+ in one case, showing ring chromosomes by FISH analysis. TP53 mutations are confirmed to be closely correlated with NR-DD liposarcomas and no CDK4 involvement was found in the myxoid/round cell liposarcoma group. As well as confirming the synergistic effect of MDM2 and CDK4, these results are consistent with the concept that amplicon(s) excluding MDM2 may contribute to transformation and support a role of CDK4 in opposing p53 function, particularly in NR WD liposarcoma.
Assuntos
Cromossomos Humanos Par 12 , Quinases Ciclina-Dependentes/genética , Amplificação de Genes , Lipossarcoma/genética , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Southern Blotting , Quinase 4 Dependente de Ciclina , Regulação da Expressão Gênica , Genes p53 , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Proteínas Proto-Oncogênicas c-mdm2RESUMO
Recent preclinical and clinical data suggest that TP53 status and TP53 mutations may be important in determining tumour aggressiveness and therapy response. In this study we investigate the feasibility of a structural and quantitative analysis of TP53 on fine-needle aspiration (FNA) material obtained from 31 consecutive female patients with breast carcinoma, enrolled in a primary chemotherapy protocol. Tumours were screened for p53 protein overexpression and TP53 mutations (exons 5-8) using immunocytochemistry, polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing analyses, and finally using fluorescence in situ hybridization (FISH) analysis. Positive nuclear staining was identified in six cases whereas mutations were detected in nine. Although the immunoreactive pattern fitted fully with the characterized TP53 mutation type, the considerable number of null p53 mutations (i.e. four) coupled with the lack of information regarding the localization of TP53 mutations make immunocytochemistry an inadequate indicator of TP53 function deregulation. Combining molecular and FISH analyses, we detected three cases with TP53 deletion and one case with deletion and mutation. Finally, DNA static-image analysis performed on 29 cases showed aneuploidy in 26 cases, which included all TP53-mutated cases. The present results show that FNA may assist clinical decisions by allowing the evaluation of a variety of biological parameters relevant for prognosis and treatment planning.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , DNA de Neoplasias/análise , Perda de Heterozigosidade/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Biópsia por Agulha , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Éxons/genética , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Mutação , Proteína Supressora de Tumor p53/análiseRESUMO
Mechanical and histochemical characteristics of the lateral gastrocnemius (LG) muscle of the rat were examined 21 days after capsaicin injection into the LG muscle. The capsaicin caused a decrease in generation rate of twitch and tetanic tension and an increase in fatigue resistance of LG muscle. The histochemical muscle fiber profile evaluated by myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide tetrazolium reductase methods showed an increase of type I and IIC fibers and a decrease of the type IIB in whole muscle, and a decrease of the IIA, IIX fibers in the red part accompanied by their increase in the white part. Therefore the capsaicin treatment, which selectively eliminated fibers belonging to the III and IV groups of muscle afferents, induced muscle fiber transformation from fast contracting fatiguing fibers to slowly contracting nonfatiguing ones.
Assuntos
Capsaicina/farmacologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculos/inervação , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Vias Aferentes/fisiologia , Animais , Denervação , Membro Posterior , Histocitoquímica , Injeções , Masculino , Músculos/efeitos dos fármacos , Músculos/fisiologia , Ratos , Ratos WistarRESUMO
Recent findings have indicated that TP53 inactivation in sarcomas may result from mutation and/or deletion of the TP53 gene or, alternatively, from binding to the MDM2 gene products. To investigate further a possible role of the two genes in sarcomas, 24 large and deep-seated lipomas and 74 liposarcomas of various subtypes were analysed for mdm2 and p53 overexpression by immunocytochemistry. Nineteen cases of the same series were also molecularly analysed for both MDM2 gene amplification and TP53 mutations, and a further ten cases for non-random chromosomal abnormalities. In the retroperitoneal well-differentiated-dedifferentiated (WD-DD) group, 15/16 WD and 8/8 DD liposarcomas displayed the mdm2+/p53+ phenotype, consistent with MDM2 gene amplification in the absence of TP53 mutations. In the non-retroperitoneal WD-DD group, 5/11 WD liposarcomas also retained the mdm2+/p53+ phenotype whereas all DD liposarcomas showed an immunophenotype and, when assessed, a genotype consistent with mutant TP53. Null mdm2 immunophenotype, coupled with evidence of a specific chromosome translocation t(12;16), was constantly observed in both the usual and the cellular subtypes of myxoid liposarcoma, three cases of which also showed TP53 alterations at the genetic or protein level. Neither mdm2 nor p53 overexpression was observed in the lipomas. The results show the existence of three main pathogenetically distinct groups of liposarcoma. The first retroperitoneal WD-DD group, which represents a novel class of tumours within a single histological category of sarcoma, where MDM2-mediated inactivation of p53 could be related to the pathogenetic mechanism. The second is the non-retroperitoneal WD-DD group, where the TP53 mutations appear to correlate with the dedifferentiation process. The third is the myxoid group, which is characterized by its own unique cytogenetic profile and never shows any involvement of TP53 or MDM2 genes. As for diagnostic significance, the absence of mdm2 and p53 reactivity in lipomas seems to represent a useful marker for differential diagnosis from lipoma-like WD liposarcomas.
Assuntos
Lipossarcoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Idoso , Aberrações Cromossômicas , Feminino , Expressão Gênica , Genótipo , Humanos , Técnicas Imunoenzimáticas , Imunofenotipagem , Lipoma/genética , Lipoma/metabolismo , Lipossarcoma/genética , Lipossarcoma/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-mdm2 , Neoplasias Retroperitoneais/genética , Neoplasias Retroperitoneais/metabolismo , Estudos RetrospectivosRESUMO
Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Genes p53 , Mutação Puntual , Etídio , Feminino , Corantes Fluorescentes , Genoma Humano , Humanos , Desnaturação de Ácido Nucleico , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Porosidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The entry pathway and central distribution of A delta and C muscle afferents within the central nervous system (CNS) were investigated by combining electron microscopy and electrophysiological analysis after intramuscular injection of capsaicin. The drug was injected into the rat lateral gastrocnemius (LG) and extraocular (EO) muscles. The compound action potentials of LG nerve and the evoked field potentials recorded in semilunar ganglion showed an immediate and permanent reduction in A delta and C components. The morphological data revealed degenerating unmyelinated axons and terminals in the inner sublamina II and in the border of laminae I-II of the dorsal horn at L4-L5 and C1-C2 (subnucleus caudalis trigemini) spinal cord segments. Most degenerating terminals were the central bouton (C) of type I and II synaptic glomeruli. Furthermore, degenerating peripheral axonal endings (V2) presynaptic to normal C were found. Since V2 were previously found degenerated after cutting the oculomotor nerve (ON) or L4 ventral root, we conclude that some A delta and C afferents from LG and EO muscles entering the CNS by ON or ventral roots make axoaxonic synapses on other primary afferents to promote an afferent control of sensory input.
Assuntos
Capsaicina/farmacologia , Gânglios/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Medula Espinal/ultraestrutura , Animais , Estimulação Elétrica , Masculino , Microscopia Eletrônica , Ratos , Ratos WistarRESUMO
The p53 protein is a multifunctional transcriptional regulator involved in cellular response to DNA damage and has been implicated as a putative determinant of sensitivity of tumor cells to cytotoxic agents. Since the p53 gene becomes inactivated in over one-half of advanced ovarian carcinoma, in this study we have examined the relationships between p53 gene alterations, p53 immunoreactivity, and response to cisplatin-based chemotherapy in ovarian cancer patients. All patients had advanced (FIGO stage III or IV) ovarian carcinoma and, with one exception, were untreated at the time of collection of tumor specimens. After initial debulking surgery, patients received high-dose cisplatin therapy. Tumor samples were analyzed for p53 gene mutations and for p53 protein accumulation, and the findings were correlated with tumor responsiveness. Of the 33 tumors examined, p53 gene mutations were found in 20 cases, including 15 missense mutations, 2 deletions, 2 nonsense mutations, and a base substitution at splice site. Twenty tumors showed positive immunostaining for p53. Only missense mutations were associated with positive immunostaining. In addition, p53 overexpression was detected in five tumors in the absence of mutations. Most (12 of 14) of the missense mutations associated with p53 protein stabilization were found refractory to therapy, as well as tumors overexpressing wild-type p53 (4 of 5). A significant correlation has been found between p53 accumulation, type of mutation (i.e., missense mutations), and pathological response to cisplatin-based therapy. In conclusion, the present results are consistent with a role of p53 as a determinant of chemosensitivity of ovarian carcinoma.
Assuntos
Cisplatino/uso terapêutico , Genes p53 , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Mutação Puntual , Deleção de Sequência , Proteína Supressora de Tumor p53/biossíntese , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Códon , Éxons , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Íntrons , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgiaRESUMO
Twenty-nine samples from 28 cases of vulvar squamous cell carcinoma, of which 13 fulfilled the criteria of the bowenoid subtype (mean age 45 years, range 31-68) and 16 of the usual subtype of invasive squamous cell carcinoma (ISCC) (mean age 67.5 years, range 34-83) were investigated for human papillomavirus (HPV) DNA, TP53 alterations, and mdm2 and bcl-2 gene product deregulation. Microscopically all the bowenoid subtype cases (group I) showed a high-grade intraepithelial (VIN 3, carcinoma in situ) lesion associated with early invasive carcinoma in six cases and overt invasive carcinoma in one. By contrast, no evidence of early carcinoma was present in the ISCCs (group II). By in situ hybridization and/or Southern blot hybridization or polymerase chain reaction (PCR), HPV DNA was detected in all cases of group I and in four of 16 cases (25%) of group II, two only by Southern blot after PCR. By single-strand conformation polymorphism and immunocytochemistry only wild-type TP53 and absence of detectable p53 product, respectively, were found in all cases of group I, i.e., in high-risk HPV-positive carcinomas, whereas mutations and/or p53 overexpression accounted for 75% in group II, i.e., in mainly HPV-negative carcinomas. The TP53 gene mutations observed in invasive carcinomas were significantly related to node-positive cases (p = 0.04). Taken together and in agreement with in vitro data, these results support the view that an alteration of TP53, gained either by interaction with viral oncoproteins or by somatic mutations, is a crucial event in the pathogenesis of vulvar carcinomas, but that TP53 mutations are mainly associated with disease progression. Finally, a preliminary immunocytochemical analysis seems to speak against the possible involvement of both MDM2 and BCL-2 gene products in the development of vulvar carcinoma.
Assuntos
Carcinoma de Células Escamosas/etiologia , Genes p53/genética , Papillomaviridae , Infecções por Papillomavirus/complicações , Infecções Tumorais por Vírus/complicações , Displasia do Colo do Útero/etiologia , Neoplasias Vulvares/etiologia , Adulto , Idoso , Sequência de Bases , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/secundário , DNA Viral/análise , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Estadiamento de Neoplasias , Infecções por Papillomavirus/patologia , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas/análise , Proteína Supressora de Tumor p53/análise , Infecções Tumorais por Vírus/patologia , Neoplasias Vulvares/química , Neoplasias Vulvares/patologia , Displasia do Colo do Útero/química , Displasia do Colo do Útero/patologiaRESUMO
The role of GABA in NMDA-dependent long term depression (LTD) in the medial vestibular nuclei (MVN) was studied on rat brainstem slices. High frequency stimulation (HFS) of the primary vestibular afferents induces a long lasting reduction of the polysynaptic (N2) component of the field potentials recorded in the dorsal portion of the MVN. The induction but not the maintenance of this depression was abolished by AP5, a specific blocking agent for glutamate NMDA receptors. The involvement of GABA in mediating the depression was checked by applying the GABAA and GABAB receptor antagonists, bicuculline and saclofen, before and after HFS. Under bicuculline and saclofen perfusion, HFS provoked a slight potentiation of the N2 wave, while the N2 depression clearly emerged after drug wash-out. This indicates that GABA is not involved in inducing the long term effect, but it is necessary for its expression. Similarly, the LTD reversed and a slight potentiation appeared when both drugs were administered after its induction. Most of these effects were due to the bicuculline, suggesting that GABAA receptors contribute to LTD more than GABAB do. According to our results, it is unlikely that the long lasting vestibular depression is the result of a homosynaptic LTD. On the contrary, our findings suggest that the depression is due to an enhancement of the GABA inhibitory effect, caused by an HFS dependent increase in gabaergic interneuron activity, which resets vestibular neuron excitability at a lower level.
Assuntos
Potenciação de Longa Duração/efeitos dos fármacos , N-Metilaspartato/farmacologia , Núcleos Vestibulares/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Bicuculina/farmacologia , Tronco Encefálico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
As previously demonstrated, high frequency stimulation (HFS) of the primary vestibular afferents always induces a clear, long lasting depression of the polysynaptic (N2) component of the field potentials recorded in the dorsal portion of the medial vestibular nuclei (MVN). The induction of the HFS effect was mediated by the activation of glutamate NMDA receptors, since it was blocked by AP5. The mechanisms at the basis of such a depression were studied. Our results demonstrate that Gaba, acting on both GabaA and GabaB receptors, is involved in mediating this phenomenon. In fact, HFS applied during Bicuculline and Saclofen perfusion, was no longer able to induce an N2 depression, but provoked a slight potentiation. However, the N2 depression clearly emerged after drug wash-out. Furthermore, Bicuculline and Saclofen fully abolished the N2 depression and highlighted the potentiation, when administered after HFS. The possibility that the N2 depression is the result of a homosynaptic LTD can be excluded on the basis of our results. On the contrary, our findings suggest that the depression is due to an enhancement of the Gaba inhibitory effect due to an HFS dependent increase in gabaergic interneuron activity, which resets vestibular neuron excitability at a lower level.
Assuntos
Inibição Neural/fisiologia , Discriminação da Altura Tonal/fisiologia , Transmissão Sináptica/fisiologia , Núcleos Vestibulares/fisiologia , Ácido gama-Aminobutírico/fisiologia , Vias Aferentes/fisiologia , Animais , Mapeamento Encefálico , Técnicas de Cultura , Interneurônios/fisiologia , Ratos , Receptores de GABA-A/fisiologia , Receptores de GABA-B/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
The injection of capsaicin into the lateral gastrocnemius (LG) muscle of the rat induced an immediate and sustained reduction in the A delta and C components of the compound action potential (CAP) of the LG motor nerve. Conversely, the drug did not immediately affect the CAP wave belonging to fast-conducting fibers or the motor responses to LG nerve stimulation. It seems that capsaicin only affects the group III and IV afferents of LG nerve. However, a week after the injection the capsaicin also altered the motor responses, as shown by the threshold enhancement and amplitude reduction of the muscle twitch and by the decrease of the A alpha-beta CAP components. This late motor impairment was attributed to a central depression following a reduction of capsaicin-sensitive neuron input into the CNS. However, this motor effect was transient since the LG nerve regained the preinjection excitability level in a week and the muscle twitch amplitude reached the control value in a month.