RESUMO
TGF-beta1 is a multifunctional regulatory peptide (25 kDa) inducing growth arrest and apoptosis in many normal and neoplastic cells. In the present study, the involvement of proapoptotic (bax) and antiapoptotic (bcl-2) genes in the molecular mechanism of TGF-beta1-induced apoptosis of L1210 leukemic cells was investigated. Bax transcript was measured using the RT-PCR method with GAPDH as a "housekeeping gene" control, whereas Bcl-2 protein was determined using flow cytometry (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated mouse anti-IgG1 antibody as a negative control). Apoptosis was evaluated using fluorescence microscopy and flow cytometry after cell staining with DAPI and sulforhodamine or propidium iodide and Hoechst 33342. ROS generation was assessed by flow cytometry using the oxidation-sensitive fluorescent marker C-DCDHF-DA. The response of L1210 leukemic cells to TGF-beta1 was two-directional: 1) partial arrest of the cell cycle at G1-S transition, and 2) induction of apoptotic cell death. TGF-beta1 increased the number of leukemic cells with typical morphological features of apoptosis: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis. DNA cleavage led to a decrease of the nuclear DNA content and the appearance of a hypodiploid peak sub-G1 in the DNA histogram. The extraction of low-molecular weight DNA fragments from apoptotic cells showed that TGF-beta1-induced apoptosis concerned first of all the cells from G1 phase. Two phases of intracellular ROS generation in TGF-beta1-treated cultures were observed: the first (rapid, 60 min after TGF-beta1 administration), and the second (slow, occurring between 24 and 48h of experiment, respectively). The increase of apoptotic cell number in TGF-beta1-treated cultures (2% FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2, and with a significant drop of Bcl-2 level in the remaining cells after 24 h. The dose-dependent relationship between TGF-beta1 concentration and Bcl-2 level was nonlinear and described by power series regression. The lowest Bcl-2 level was noted at 1 ng/ml of TGF-beta1 concentration. The increase of Bax mRNA:GAPDH mRNA ratio was observed 3h after TGF-beta1 (1 ng/ml) administration to both the maintenance (2% FCS/RPMI) and growth promoting (10% FCS/RPMI) medium. Regardless of TGF-beta1 treatment, the quantity of Bax transcript was dependent on FCS concentration, being higher in the growth promoting medium. The results of this study indicate that bax may function as a primary response gene and together with lowered Bcl-2 level may determine the induction of apoptotic process in L1210 leukemic cells exposed to TGF-beta1.
Assuntos
Apoptose/genética , Leucemia L1210/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Leucemia L1210/patologia , Camundongos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2RESUMO
The expression of apoptosis-related proteins: TGF-beta1 (local inductor), TGF-beta-receptor, Bax (promoter), Bcl-2 (inhibitor) and CPP-32 (executor of apoptosis); the subcellular distribution of Bax; as well as the number and morphology of apoptotic cells in low-, moderate-, and high-involuted mammary glands of sow (four to six days after weaning) were investigated. The immunohistochemical study demonstrated a statistically significant increase in the integrated optical density (IOD) of lobuloalveolar mammary tissue labelling with anti-Bax antibody from low- through moderate-, to high-involuted glands. The immunoelectron microscopy revealed that Bax was localised in the cytosol, on the membranes of mitochondrium and rough endoplasmic reticulum, in nuclear envelope pores, and over heterochromatin of mammary epithelial cells. The increase in Bax/Bcl-2 ratio (2.3, 2.6 and 5.6 for low-, moderate-, and high-involuted glands, respectively) indicated the increasing susceptibility of mammary epithelial cells to apoptosis in the course of involution. The highest Bax/Bcl-2 ratio in high-involuted glands coincided with the highest expression of CPP-32 (caspase 3), TGF-beta1 and TGF-beta1 receptor. The number of apoptotic cells (simultaneous TUNEL and Hoechst 33342 staining) was 2.7, 3.4 and 3.8% for low-, moderate-, and high-involuted glands, respectively. The ultrastructural evaluation showed characteristic morphological features of apoptosis such as: margination and condensation of chromatin; pyknosis and fragmentation of the nucleus; and formation of apoptotic bodies. Phagocytosis of apoptotic cells by macrophages was also documented. The results of the present study suggest the involvement of Bax/Bcl-2 check-point in the regulation, CPP-32 in the execution, but TGF-beta1 in the induction of apoptosis of mammary epithelial cells in the involuting mammary gland of sow.
Assuntos
Apoptose/efeitos dos fármacos , Glândulas Mamárias Animais/citologia , Suínos/metabolismo , Animais , Caspase 3 , Caspases/metabolismo , Caspases/farmacologia , Células Epiteliais/fisiologia , Feminino , Modelos Animais , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Desmame , Proteína X Associada a bcl-2RESUMO
The expression of apoptosis-related proteins: TGF-beta1 (auto/paracrine inducer) and its receptor (TGF-betaRIII), Bax (promoter), Bcl-2 (inhibitor) and CPP-32 (executor of apoptosis) as well as the apoptotic cell number in mammary glands of 11 Polish White Improved goats in the course of the lactation cycle (peak of lactation: days 40-70, late lactation: days 208-256, drying off: days 267-340) was investigated. The immunohistochemical study demonstrated a significant increase in TGF-beta1 and TGF-betaRIII expression in the lobuloalveolar tissue from the early lactation to the dry period. Our recent study on HC11 mouse mammary epithelial cells [Cell. Mol. Biol. 46 (2000) 175] has revealed an inhibitory effect of prolactin on TGF-beta1 transcription, which may explain the low TGF-beta1 synthesis during lactogenesis and galactopoiesis and the increase in TGF-beta1 and TGF-betaIIIR expression in late lactation and dry period. Bax expression was the lowest in the peak of lactation, significantly increased in late lactation and remained elevated during drying off. Bcl-2 content was lower than Bax in all examined periods, but it increased significantly at the end of lactation, which suggests the survival of cells with the highest resistance to apoptogenic stimuli. The increase in Bcl-2 level in remnant lobuloalveolar tissue is probably the molecular mechanism that limits the rate of secretory tissue involution. The induction of CPP-32 (caspase 3) from the peak of lactation to dry period was accompanied by a progressive loss of mammary epithelial cells and the increase in apoptotic cell numbers but only in the dry period. The increase in the expression of examined proteins in the late lactation and the dry period indicates their involvement in the induction (TGF-beta1 and TGF-betaRIII), regulation (Bax and Bcl-2) and execution (CPP-32) of programmed cell death in the course of mammary gland involution. The lack of an increase in apoptotic cell number in late lactation, in spite of the evident decrease in total cell number, suggests milk as an alternative route (apart from phagocytosis) of apoptotic cells elimination from the mammary gland. The presented results provide new insights into the molecular mechanism of mammary cell apoptosis in goat and for this reason may have practical implications for control and regulation of mammary gland remodelling, which is a prerequisite for subsequent successful lactation.
RESUMO
BAX is the 192-amino acid, 21-kDa protein which is ubiquitously distributed in normal tissues and is regarded as a tumor suppressor sensitizing malignant cells to anticancer drugs. In spite of many studies, the molecular mechanism of BAX action is still obscure. In the present study subcellular BAX translocations in human colon adenocarcinoma COLO 205 cells exposed to various anticancer drugs [camptothecin (CPT), etoposide (ETO), staurosporine (STP), 2-chloro-2'-deoxyadenosine (2CdA) and nimesulide (NIM)] was examined. Cells were grown on coverslips under optimal conditions (10% FCS/DMEM) or were stimulated to apoptosis with the drugs examined. Laser scanning cytometry was applied for the quantitative analysis of BAX expression, and distribution in the cytoplasmic (BAX Cf) and nuclear (BAX Nf) area. BAX maximal pixel (BAX MP), the parameter corresponding to aggregation of BAX in the cell, was also measured. All examined drugs increased the number of cells with high BAX MP, reaching the peak at 60 min after drug administration. The most pronounced effect was in the case of 2CdA, CPT and STP. The increase in BAX MP was observed only when antibody recognizing the 43-61 amino acid sequence was used. When antibody binding the N-terminal epitope (11-30 amino acid sequence) was applied, the number of cells expressing high BAX MP significantly decreased. These results indicate that apoptotic stimuli delivered by anticancer drugs led to aggregation of BAX in cancer cells, which is dependent on BAX activation by its cleavage at the N-terminal epitope and exposure of the BH3 domain. It was shown that BAX Nf increased in cells treated with CPT, STP, ETO, 2CdA and NIM, whereas BAX Cf rose after STP and NIM. The increase in BAX Nf and, occurring in most treatments, the increase in the BAX Nf:Cf ratio indicates a BAX shift from the cytoplasm to the nucleus. Furthermore, staining with different antibodies showed that only the activated form of BAX was translocated to the nucleus. Immunoelectron microscopy revealed that CPT-induced apoptosis was associated with translocation of BAX from the cytosol to organellar membranes (mitochondrial, Golgi apparatus and endoplasmic reticulum) and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. The subcellular translocations of BAX preceded in time the appearance of morphological symptoms of apoptosis. In conclusion, (i) in spite of different molecular mechanisms of apoptosis induction by the anticancer drugs examined, BAX remains a common link in the chain of reactions leading to cell death, and (ii) BAX activation and subcellular translocations from the cytosol to organellar membranes and nucleus are key cellular responses to drugs bearing proapoptotic properties.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células Tumorais Cultivadas/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Western Blotting , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2 , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Isoenzimas/antagonistas & inibidores , Proteínas de Membrana , Microscopia Imunoeletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Prostaglandina-Endoperóxido Sintases , Inibidores da Topoisomerase I , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , Proteína X Associada a bcl-2RESUMO
In this study we wanted to determine whether changes in antioxidant profile could follow the catabolic effects of glucocorticoids. We also wanted to compare resistance to glucocorticoid overload in young and old rats. To address these questions, whole body catabolism was induced by the administration of dexamethasone (Dex) at either 2 mg/kg bodyweight/day to young (6 weeks old) or 0.5 mg/kg body-weight/day to old (94 weeks old) rats. Bodyweight loss of pair-fed rats not given Dex was only 2% in the young rats and 8% in the old rats, whereas in Dex-treated rats the decrease in bodyweight was 22% in the young rats and 13% in the old rats after 5 days of treatment. Spleen weight decreased by 65% in the young rats and by 52% in the old rats. Additionally, in the young rats there was a 46% reduction in glutathione (GSH) in erythrocytes as well as a 36% reduction in GSH/tissue wet weight in the soleus muscle. The corresponding figures for the old rats were 35 and 26%, respectively. Taken together, these results suggest that Dex directly and/or indirectly impaired the antioxidant reactions. This was further confirmed by a significant (50%) decline in Cu-Zn superoxide dismutase (SOD-1) activity in erythrocytes isolated from the young rats treated with Dex but not the old rats as they showed a significant elevation in SOD-1 activity (by 101%). Thiobarbituric acid reactant substances were significantly higher in both young and old rats. Activity of blood plasma creatine kinase increased by 73% in the young rats and by 307% in the old rats treated with Dex. Although both the young and the old rats could recover from oxidative stress, the old rats in contrast to the young rats remained catabolic until the end of the experiment. In conclusion, we suggest that old rats are more vulnerable to the catabolic action of Dex, whereas young rats are more susceptible to the oxidative stress induced by Dex.
Assuntos
Antioxidantes/metabolismo , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Músculo Esquelético/metabolismo , Estresse Oxidativo , Baço/patologia , Fatores Etários , Animais , Animais Recém-Nascidos , Atrofia , Creatina Quinase/sangue , Glutationa/sangue , Glutationa/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Tamanho do Órgão , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Superóxido Dismutase/sangue , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
The effect of TGF-beta1, an auto/paracrine antiproliferative and apoptogenic factor on Bax transcript level (RT-PCR), subcellular distribution of Bax protein (immunoelectron microscopy), Bcl-2 protein level and apoptotic cell number (flow cytometry with FITC-conjugated monoclonal anti-Bcl-2 antibody and DNA stained with DAPI) in HC11 mouse mammary epithelial cells was examined. TGF-beta1 increased Bax transcript level (evaluated by Bax mRNA/GAPDH mRNA ratio) and stimulated Bax protein movement from cytosol to organellar membranes, mainly mitochondrial, during 60 min. The new observation is the presence of Bax on channel membranes of Golgi apparatus and translocation of Bax from cytosol to the fibrous nucleoplasm via nuclear envelope pores (especially after 120 min. of cell exposure to TGF-beta1). Prolactin protected HC11 cells against TGF-beta1-induced PCD, which could occur at two levels: 1) TGF-beta1 expression, through the decrease of TGF-beta1 transcript content, and 2) Bax/Bcl-2 checkpoint, through down-regulation of Bax and up-regulation of Bcl-2. In conclusion, Bax and Bcl-2 proteins are implicated in the mechanism of TGF-beta1-induced PCD and antiapoptotic action of prolactin in HC11 mouse mammary epithelial cells. The activation of transcription and redistribution of Bax from cytosol to organellar membranes and nucleus constitute the early events in the cellular response to TGF-beta1.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Contagem de Células/efeitos dos fármacos , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Microscopia Imunoeletrônica , Prolactina/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Proteína X Associada a bcl-2RESUMO
The effect of prolactin on apoptosis and the expression of bcl-2 and bax in HC11 mouse mammary epithelial cells were investigated. Flow cytometric analysis of Bcl-2 level (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated monoclonal anti-IgG1 antibody as a negative control), number of apoptotic cells and cell cycle phases (DNA stained with DAPI) was performed. Bax transcript was measured using the RT-PCR method with GAPDH serving as a reference gene. Administration of prolactin (5 microg/ml) in the presence of insulin stimulated differentiation of mammary epithelial cells, which manifested in stopping cells at G0/G1 phase, cell swelling and increase of cell number with enhanced protein content. Moreover, prolactin highly significantly reduced the extent of apoptosis of HC11 cells during 48 h of incubation. Nevertheless, the apoptotic cell number rose with increased time length of cell culture, probably due to the resulting high cell density and EGF withdrawal from t he incubation medium. The antiapoptotic effect of prolactin was associated with up-regulation of bcl-2 expression, shown as an increase in cell numbers expressing this protooncogene and elevated Bcl-2 content in these cells. A negative relationship (r=-0.87, p< or =0.001) between the number of apoptotic cells and those expressing bcl-2 was also found. Prolactin administration lowered Bax transcript by 68.8% and 70.7% after 3 and 6h, respectively. In conclusion, the results presented indicate that stimulation of bcl-2 expression with simultaneous suppression of bax may be key events in the mechanism of antiapoptotic action of prolactin in HC11 mammary epithelial cells.