RESUMO
BACKGROUND: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour spread and metastasis; nm23 is a promising candidate for a metastasis suppressing gene. AIMS: To evaluate whether expression of nm23-H1 protein or loss of heterozygosity (LOH) of the nm23-H1 gene is associated with colon cancer progression. MATERIALS/METHODS: Paraffin wax embedded tissue sections were analysed immunohistochemically. DNA isolated from normal and tumour tissue was used for LOH analysis using a variable nucleotide tandem repeat (VNTR) marker located in the untranslated 5' region of the nm23-H1 gene. RNA isolated from tumour and normal tissue was used for "real time" RT-PCR. RESULTS: Of 102 adenocarcinomas examined, 58.8% stained weakly for nm23-H1 protein. There was a negative correlation between nm23-H1 positivity and tumour histological grade. In VNTR analysis, 70.2% of patients were informative and 27.4% of tumours had nm23-H1 LOH. There was a positive correlation between nm23-H1 LOH and both tumour histological grade and Dukes's stage. Expression of nm23-H1 mRNA was increased in 22 of 30 colon tumours compared with normal tissue. No significant correlation was found between nm23-H1 mRNA expression and histological grade or Dukes's stage of tumours. CONCLUSIONS: These findings suggest that nm23-H1 protein expression in early stages may have a role in suppressing metastasis in sporadic colon cancer, whereas at a later stage both reduced nm23-H1 protein expression and LOH of the nm23-H1 gene may play role in colon cancer progression and metastasis.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Perda de Heterozigosidade/genética , Núcleosídeo-Difosfato Quinase/análise , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/análise , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , DNA de Neoplasias/genética , Feminino , Genes Supressores de Tumor/fisiologia , Humanos , Imuno-Histoquímica/métodos , Masculino , Nucleosídeo NM23 Difosfato Quinases , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Neoplásico/análise , Análise de SobrevidaRESUMO
Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 micromol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Compostos Organometálicos/metabolismo , Fenantrolinas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Vanádio/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Fosfatase 1 , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The purpose of this study was to examine the short-term effects of 75, 100 and 150 mg of acarbose mixed in 100 g standard laboratory chow on specific intestinal disaccharidase activities and on hyperglycaemia in diabetic CBA strain mice on standard diet. The small intestine was excised and divided into three segments, from pylorus to duodenum, and two equal lengths of the jejunum and ileum of control and diabetic mice with or without added acarbose. Specific maltase and sucrase activities were determined using maltose and sucrose as substrates respectively. Increased specific activities of maltase and sucrase were detected in the intestines of CBA mice on standard laboratory diet seven days after alloxan-induced diabetes. Feeding for 7 days with 75, 100 or 150 mg acarbose uniformly mixed in 100 g standard laboratory chow, induced a decrease in the specific maltase and sucrase activities, compared with diabetic mice on standard laboratory diet. Feeding with 75 mg acarbose mixed in 100 g standard laboratory chow caused a statistically significant decrease of maltase in the duodenum and of sucrase in duodenum and jejunum, without a antihyperglycaemic effect. Feeding with 100 or 150 mg caused statistically significant decreases in specific maltase and sucrase activities in duodenum, jejunum and ileum. An antihyperglycaemic effect was observed only in the group of diabetic mice fed with 100 mg acarbose. This indicates that the antihyperglycaemic effect of acarbose involves factors other than these, related only to its inhibitory effect on disaccharidase activities.
Assuntos
Acarbose/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Glicosídeo Hidrolases , Hiperglicemia/tratamento farmacológico , Intestinos/enzimologia , Sacarase/antagonistas & inibidores , Acarbose/uso terapêutico , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/uso terapêutico , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos CBA , Distribuição Aleatória , Sacarase/metabolismo , alfa-Glucosidases/metabolismoRESUMO
Changes in the concentration of glutathione S-transferases (GSTs) and malondialdehyde (MDA) were assessed in the liver of normal and diabetic NOD mice with and without treatment with the plant extract P-9801091. The plant extract P-9801091 is an antihyperglycaemic preparation containing Myrtilli folium (Vaccinium myrtillus L.), Taraxaci radix (Taraxacum of fi cinale Web.), Cichorii radix (Cichorium intybus L.), Juniperi fructus (Juniperus communis L.), Centaurii herba (Centaurium umbellatum Gilib.), Phaseoli pericarpium (Phaseolus vulgaris L.), Millefoliiherba (Achillea millefolium L.), Mori folium (Morus nigra L.), Valerianae radix (Valeriana of ficinalis L.) and Urticae herba et radix (Urtica dioica L). Hyperglycaemia in diabetes mellitus is responsible for the development of oxidative stress (via glucose auto-oxidation and protein glycation), which is characterized by increased lipid peroxide production (MDA is a lipid peroxidation end product) and/or decreased antioxidative defence (GST in the liver is predominantly an alpha enzyme, which has antioxidative activity). The catalytic concentration of GSTs in the liver was significantly reduced in diabetic NOD mice compared with normal NOD mice (p < 0.01), while the concentration of MDA showed a rising tendency (not significant). The results showed that statistically significant changes in antioxidative defence occurred in the experimental model of short-term diabetes mellitus. A 7-day treatment with P-9801091 plant extract at a dose of 20 mg/kg body mass led to a significant increase in the catalytic concentration of GSTs in the liver of diabetic NOD mice (p < 0.01) and a decrease in MDA concentration (not significant), which could be explained by its antihyperglycaemic effect.
Assuntos
Hipoglicemiantes/farmacologia , Fígado/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/farmacologia , Plantas Medicinais , Animais , Glutationa Transferase/efeitos dos fármacos , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/uso terapêutico , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêuticoRESUMO
BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.
Assuntos
Hidrolases Anidrido Ácido , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteínas de Neoplasias/genética , Adulto , Idoso , Apoptose/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Divisão Celular/genética , Fragilidade Cromossômica/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , DNA de Neoplasias/genética , Feminino , Expressão Gênica , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , PrognósticoRESUMO
Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.
Assuntos
Adjuvantes Farmacêuticos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases , Zeolitas/uso terapêutico , Adjuvantes Farmacêuticos/efeitos adversos , Adjuvantes Farmacêuticos/farmacologia , Envelhecimento/fisiologia , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proteínas de Ciclo Celular/análise , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/análise , Doenças do Cão/tratamento farmacológico , Doenças do Cão/patologia , Cães , Feminino , Células HeLa , Humanos , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neoplasias/patologia , Neoplasias/veterinária , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/análise , Zeolitas/efeitos adversos , Zeolitas/farmacologiaRESUMO
We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.
Assuntos
Neoplasias do Colo/genética , Proteínas de Ligação a DNA/genética , Transativadores/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Genes Supressores de Tumor , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Smad4RESUMO
The antihyperglycemic effect of the Antidiabetis herbal preparation ((Myrtilli folium (Vaccinium myrtillus L.), Taraxaci radix (Taraxacum officinale Web.), Cichorii radix (Cichorium intybus L.), Juniperi fructus (Juniperus communis L.), Centaurii herba (Centaurium umbellatum Gilib.), Phaseoli pericarpium (Phaseolus vulgaris), Millefollii herba (Achillea millefolium L.), Morii folium (Morus nigra L.), Valeriane radix (Valleriana officinalis L.), Urticae herba et radix (Urtica dioica L.)), patent No. P-9801091 Zagreb, Croatia was investigated. Two extracts were prepared: ethanol extract (extract 1), and ethanol extract from which ethanol was evaporated on a rotatory evaporator at a temperature of 45 degrees C (extract 2). Extract 1 and extract 2 were administered (in experiment 1) to alloxan-induced non-obese diabetic (NOD) mice in the same dose of 20 mg/kg. Blood glucose was determined before, and 10, 30, 60 and 120 min after the preparation administration. Extract 1 and extract 2 decreased the level of blood glucose by 10 and 20%, respectively, of the initial value (at 0 min, mean = 22.6 +/- 8.3 mmol/l). Serum levels of glucose and fructosamine were determined in NOD mice, NOD mice administered extract 2 in a dose of 20 mg/kg of extract 2, and NOD mice administered acarbose in a dose of 25 mg/100 g chow, in order to verify the hypoglycemic action of extract 2 (in experiment 2). Extract 2 and acarbose were admixed to the chow. The duration of treatment was 7 days. Significantly lower glucose (P < 0.05) and fructosamine (P < 0.001) levels were recorded in extract 2 treated NOD mice as compared with NOD mice. Study results showed extract 2 to significantly decrease the level of glucose and fructosamine in alloxan induced NOD mice. Our future studies will be focused on the search of active principles of the extracts.
Assuntos
Acarbose/uso terapêutico , Glicemia/análise , Diabetes Mellitus Tipo 1/tratamento farmacológico , Frutosamina/sangue , Hipoglicemiantes/uso terapêutico , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/sangue , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
We described before that iron-containing, anti-anaemic drug, ferric-sorbitol-citrate complex (FSC) inhibited proliferation of various murine cancer cells in vitro and caused tumour regression in vivo, but did not affect proliferation of the non-malignant cells. The aim of this study was to evaluate further the anticancer activity mechanism of FSC using human colon cancer cell line CaCo2. After treatment with FSC for 72 hours impaired proliferative ability and viability of CaCo2 cells as observed. Growth modification caused by FSC involved diminished expression of Bcl-2, and over-expression of mp53 proto-oncogenes, accompanied by increased incidence of apoptosis. Immunostaining the cells applying monoclonal antibodies for lipid peroxidation product 4-hydroxynonenal (HNE) showed that FSC-iron increased intracellular HNE, but did not induce severe HNE-mediated oxidative stress. Thus, antitumorous mechanism of FSC involves modulation of oncogene expression and induction of apoptosis apparently not triggered by lipid peroxidation-mediated oxidative stress, although FSC might restore endogenous HNE production in the CaCo2 cells to level resembling physiological for various non-malignant cells and tissues. Higher dose of FSC increased also number of intracellular ferritin positive CaCo2 cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácido Cítrico/farmacologia , Compostos Férricos/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Proto-Oncogenes , Sorbitol/farmacologia , Aldeídos/metabolismo , Células CACO-2 , Combinação de Medicamentos , Genes p53 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologiaRESUMO
Galectin-3 is a beta-galactoside-binding lectin that has been implicated in numerous physiological processes, including mRNA splicing, cell differentiation, tumor metastasis and the stress response. We have studied effects of transfer of resident murine peritoneal macrophages to in vitro conditions on galectin-3 in different cell compartments. Galectin-3 was purified by immunoprecipitation with rat monoclonal antibody M3/38, and analyzed by immunoblotting using the same antibody. Transfer to in vitro conditions nearly doubled the total amount of galectin-3 in cells, and caused significant alterations in its intracellular distribution, indicating that galectin-3 is involved in the adaptation of peritoneal macrophages to in vitro conditions.
Assuntos
Antígenos de Diferenciação/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Células Cultivadas , Galectina 3 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Frações Subcelulares/metabolismoRESUMO
Potassium bisperoxo(1,10-phenanthroline)oxovanadate, bpV(phen), a powerful protein phosphotyrosine phosphatase inhibitor and a potent insulinomimetic, influenced three fundamental cellular processes in HL-60 human leukemic cells: 1) inhibition of proliferation, 2) induction of differentiation and 3) apoptotic cell death. In the presence of micromolar concentrations of bpV(phen) cell number and DNA synthesis decreased progressively with time of incubation. A single treatment with bpV(phen) (3 microM) activated a differentiation program; after 6 days of incubation 82% of cells were differentiated, but differentiation started already within the first 24 h. Concentrations of 5-10 microM bpV(phen) caused the characteristic DNA ladder pattern, starting after 4.5 h. Differentiation in HL-60 cells appear to be associated with activation of extracellular signal-regulated kinase while apoptosis is connected with phosphorylation and activation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in a concentration and time-dependent manner. The antiproliferative and apoptotic action of bpV(phen) could be exploited in combination chemotherapy in leukemia.
Assuntos
Inibidores Enzimáticos/farmacologia , Células HL-60/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Fenantrolinas/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Apoptose , Western Blotting , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HL-60/citologia , Células HL-60/enzimologia , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fatores de TempoRESUMO
The aim of this study was to establish an in vitro model to confirm earlier observations on the role of the myc/ras oncogenes as promoting factors in the process of normal Langerhans islet beta cell transformation. For that purpose we infected primary mouse Langerhans islets with a recombinant retrovirus containing the v-H-ras and v-myc oncogenes, before or after treatment with transforming growth factor alpha (TGFalpha). Normal Langerhans islets, when grown in culture, are viable for 2-3 weeks. After treatment with TGFalpha, viability was extended by 10 days, following which islets disintegrated. Langerhans islets transformed with v-H-ras and v-myc became immortal and insulin negative. Single infected beta cells, liberated from a primary islet into the surrounding medium, gave rise to neo islet formation. Moreover, single infected beta cells were able to grow and divide, even without fibroblast support. These results indicate that the myc and ras oncogenes are sufficient for commencement of beta cell transformation and, therefore, could represent 'early events' in the multistep carcinogenesis of insulinomas.
Assuntos
Insulinoma/genética , Neoplasias Pancreáticas/genética , Células 3T3 , Animais , Transformação Celular Neoplásica/genética , Genes myc/fisiologia , Genes ras/fisiologia , Vetores Genéticos/administração & dosagem , Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos CBA , Retroviridae/efeitos dos fármacos , TransfecçãoRESUMO
In our previous paper we have described the isolation and characterization of a doxorubicin (DOX) resistant subline of breast adenocarcinoma SC6 cells. These cells were obtained after the treatment with low, clinically relevant doses of doxorubicin. They became cross-resistant to different wide used cytostatics. The expression of several genes involved in mitotic signal transduction, as well as cathepsins D and L, was similar in both parental and doxorubicin treated cells. The aim of this study was to examine the molecular mechanisms involved in resistance of these cells to doxorubicin. Activity of plasma membrane Pgp was examined in parental and resistant cells due to rhodamine-accumulation assay. The involvement of glutathione (GSH) and glutathione S-transferase (GST) in resistance to doxorubicin was determined in MTT modified assay due to the addition of specific inhibitors: buthionine sulfoximine (for GSH) or ethacrynic acid (for GST). The kinetic of apoptosis was followed after the treatment with DOX in control and SC6 cells by fluorescent microscope. The occurrence of apoptosis was confirmed by analysing DNA fragmentation in agarose gel. Our results indicate that P-glycoprotein, glutathione or glutathione transferases were not involved in resistance of SC6 cells to doxorubicin. However, the apoptosis was inhibited in doxorubicin-resistant cells. Therefore, even low doses of doxorubicin can induce the resistance to this drug due to inhibition of apoptosis.
Assuntos
Adenocarcinoma/patologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Fragmentação do DNA , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos , Feminino , Glutationa/análise , Glutationa Transferase/análise , Humanos , Proteínas de Neoplasias/análise , Células Tumorais CultivadasRESUMO
Myeloid leukaemia (ML) is strain specific for RFM mice, which were used in these experiments. We investigated the presence of c-myc protein during the growth of ML and after irradiation of leukaemic cells. Leukaemic spleen cells were investigated 9 (nonterminal phase NTP) or 12 days (terminal phase TP) after the injection of ML cells. Leukaemic cells of NTP were irradiated with X-rays or UV-light. c-Myc protein was detected by immunocytochemical method. c-Myc protein was expressed in 74.98% of spleen cells of healthy RFM mice. In the early period of leukaemia growth (NTP) only 14.33% of c-myc positive cells were found, as opposed to the terminal phase (TP) of leukaemia when 89.7% of c-myc positive cells were detected. These results indicated the connection of growth of ML and the presence of c-myc protein. If the spleen cells of NTP of leukaemia were irradiated by X-rays or UV-light, the number of cells which expressed c-myc protein was extremely increased.
Assuntos
Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-myc/análise , Animais , Imuno-Histoquímica , Leucemia Mieloide/patologia , Leucemia Mieloide/radioterapia , CamundongosRESUMO
We determined whether disulfide-linked insulin peptides that are immunogenic in vitro for CD4+ T cells bind to major histocompatibility complex class II in vivo. Radiolabeled recombinant human insulin (rHI) was injected into BALB/c mice, and processed rHI peptides bound to I-Ad molecules on different thymic antigen-presenting cells were characterized. The A6-A11/B7-B19 and A19-A21/B14-B21 disulfide-linked I-Ad-bound rHI peptides were isolated from thymic epithelial cells but not dendritic cells. While both thymic epithelial cells and dendritic cells present rHI to HI/I-Ad-specific T cells, these antigen-presenting cells do not present the reduced or nonreduced forms of the disulfide-linked rHI peptides. Thus, a naturally processed disulfide-linked peptide can bind to major histocompatibility complex class II in vivo. The potential role of these peptides in immunological tolerance is discussed.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Insulina/metabolismo , Timo/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Dissulfetos , Epitélio/imunologia , Humanos , Insulina/química , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/imunologia , Ligação Proteica , Proteínas Recombinantes , Timo/citologiaRESUMO
A microanalytical procedure for the determination of total and surface sialic acid concentrations was employed to establish their changes in relation to the length of alloxan diabetes in rat islets of Langerhans. 14 and 60 days after alloxan administration (65 mg/kg), the number of Langerhans islets was significantly decreased (p < 0.001) compared to the control. According to their size, the distribution of islets displayed no significant difference in diabetic and control animals 14 days after alloxan administration, while after 60 days no large islets (dia > or = 128 microns) were found in diabetic animals. The surface sialic acid was significantly increased in the small islets (p < 0.001), whereas no change was found in the large islets 14 days after alloxan administration. After 60 days, the surface sialic acid of both small and large islets was significantly decreased (p < 0.001). These results demonstrate that chronic beta-cell destruction induces a decrease in the sialic acid content in the pancreatic islet cells, suggesting that sialic acid might play a role in insulin secretory regulation regarding chronic effects of alloxan beta-cytotoxicity.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Ilhotas Pancreáticas/metabolismo , Ácidos Siálicos/metabolismo , Animais , Membrana Celular/metabolismo , Masculino , Ácido N-Acetilneuramínico , Neuraminidase/metabolismo , Neuraminidase/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The design of a specific blocking peptide for the immunosuppressive therapy of an autoimmune disease requires the identification of peptides of an autoantigen that are physiologically processed in vivo and bind to MHC-encoded membrane glycoproteins. However, knowledge of how an antigen is physiologically processed by antigen-presenting cells (APC) in vivo, particularly in the thymus, is lacking. It is also unknown whether the processing of an antigen by different APC in the thymus can influence thymic T-cell selection. This is an important consideration for attempts to delete or inactivate autoreactive T cells that elicit autoimmune disease. To address these issues, we investigated the processing of biosynthetically labelled recombinant human insulin (rHI), a model autoantigen, injected into mice and characterized the insulin peptides associated with MHC class II molecules on thymic epithelial cells and dendritic cells. These APC were found to differ in the way they process insulin. The detection of MHC-class-II-bound insulin peptides on the surface of the epithelial cells but not the dendritic cells correlated with their capacity to either present or not present insulin to T cells, respectively. Thus, antigen processing may control the appearance of different peptide-MHC class II complexes on thymic APC that mediate positive and negative selection, and thereby influence the development of the T-cell repertoire. Our findings could have important bearing on the future design of synthetic blocking peptides that reduce or eliminate the onset of autoimmune disease.
Assuntos
Autoantígenos/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Insulina/genética , Insulina/imunologia , Camundongos , Proteínas Recombinantes , Timo/imunologiaRESUMO
In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Concanavalina A/farmacologia , Diabetes Mellitus Tipo 1/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Fatores Etários , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3 , Cálcio/metabolismo , Interleucina-2/farmacologia , Ionomicina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Antígenos de Linfócitos T/análise , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The metabolism of tryptophan (TRP) was studied in diabetic and insulin-treated diabetic rats throughout a five-month period. In alloxan diabetic rats the serum and brain TRP levels were decreased (serum: 38 to 48 mmol/l, brain: 8.6 to 9.2 mmol/g) in comparison to the values of control rats (serum: 59 to 64 mmol/l, brain: 11.3 to 12.6 mmol/g). Daily long-term (for 45, 75, 90 or 135 days) treatment with intermediately acting insulin (4 IU/rat, s.c.) was not able to restore brain concentration of TRP. On the contrary, the serum TRP concentrations were totally or partially restored. The concentrations of branched chain amino acids (BCAA) were increased in serum (valine = 361.2 to 461.0 mumol/l or leucine + isoleucine = 431.0 to 520.3 mumol/l) throughout the entire five-month examination period. Insulin treatment did not return serum concentration of BCAA to normal level in the observation period either.