RESUMO
Autophagy is usually a pro-survival mechanism in cancer cells, especially in the course of chemotherapy, thus autophagy inhibition may enhance the chemotherapy-mediated anti-cancer effect. However, since autophagy is strongly involved in the immunogenicity of cell death by promoting ATP release, its inhibition may reduce the immune response against tumors, negatively influencing the overall outcome of chemotherapy. In this study, we evaluated the in vitro and in vivo anti-cancer effect of curcumin (CUR) against Her2/neu overexpressing breast cancer cells (TUBO) in the presence or in the absence of the autophagy inhibitor chloroquine (CQ). We found that TUBO cell death induced by CUR was increased in vitro by CQ and slightly in vivo in nude mice. Conversely, CQ counteracted the Cur cytotoxic effect in immune competent mice, as demonstrated by the lack of in vivo tumor regression and the reduction of overall mice survival as compared with CUR-treated mice. Immunohistochemistry analysis revealed the presence of a remarkable FoxP3 T cell infiltrate within the tumors in CUR/CQ treated mice and a reduction of T cytotoxic cells, as compared with single CUR treatment. These findings suggest that autophagy is important to elicit anti-tumor immune response and that autophagy inhibition by CQ reduces such response also by recruiting T regulatory (Treg) cells in the tumor microenvironment that may be pro-tumorigenic and might counteract CUR-mediated anti-cancer effects.
RESUMO
Hyperglycemia, the condition of high blood glucose, is typical of diabetes and obesity and represents a significant clinical problem. The relationship between hyperglycemia and cancer risk has been established by several studies. Moreover, hyperglycemia has been shown to reduce cancer cell response to therapies, conferring resistance to drug-induced cell death. Therefore, counteracting the negative effects of hyperglycemia may positively improve the cancer cell death induced by chemotherapies. Recent studies showed that zinc supplementation may have beneficial effects on glycemic control. Here we aimed at evaluating whether ZnCl2 could counteract the high-glucose (HG) effects and consequently restore the drug-induced cancer cell death. At the molecular level we found that the HG-induced expression of genes known to be involved in chemoresistance (such as HIF-1α, GLUT1, and HK2 glycolytic genes, as well as NF-κB activity) was reduced by ZnCl2 treatment. In agreement, the adryamicin (ADR)-induced apoptotic cancer cell death was significantly impaired by HG and efficiently re-established by ZnCl2 cotreatment. Mechanistically, the ADR-induced c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) phosphorylation, inhibited by HG, was efficiently restored by ZnCl2. The JNK involvement in apoptotic cell death was assessed by the use of JNK dominant-negative expression vector that indeed impaired the ZnCl2 ability to restore drug-induced cell death in HG condition. Altogether, these findings indicate that ZnCl2 supplementation efficiently restored the drug-induced cancer cell death, inhibited by HG, by both sustaining JNK activation and counteracting the glycolytic pathway.
Assuntos
Cloretos/farmacologia , Doxorrubicina/farmacologia , Glucose/toxicidade , Compostos de Zinco/farmacologia , Morte Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Desoxiglucose/metabolismo , Regulação para Baixo/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Células HCT116 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacosRESUMO
Stimulation of the Ras/MAPK cascade can either activate p53 and promote replicative senescence and apoptosis, or degrade p53 and promote cell survival. Here we show that p53 can directly counteract the Ras/MAPK signaling by inactivating ERK2/MAPK. This inactivation is due to a caspase cleavage of the ERK2 protein and contributes to p53-mediated growth arrest. We found that in Ras-transformed cells, growth arrest induced by p53, but not p21(Waf1), is associated with a strong reduction in ERK2 activity, phosphorylation, and protein half-life, and with the appearance of caspase activity. Likewise, DNA damage-induced cell cycle arrest correlates with p53-dependent ERK2 downregulation and caspase activation. Furthermore, caspase inhibitors or expression of a caspase-resistant ERK2 mutant interfere with ERK2 cleavage and restore proliferation in the presence of p53 activation, indicating that caspase-mediated ERK2 degradation contributes to p53-induced growth arrest. These findings strongly point to ERK2 as a novel p53 target in growth suppression.
Assuntos
Caspases/metabolismo , Divisão Celular/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Caspase 3 , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Dano ao DNA/fisiologia , Regulação para Baixo , Doxorrubicina/farmacologia , CamundongosRESUMO
Mitogen-activated protein kinase kinase 3 (MAP2K3, MKK3) is a member of the dual specificity protein kinase group that belongs to the MAP kinase kinase family. This kinase is activated by mitogenic or stress-inducing stimuli and participates in the MAP kinase-mediated signaling cascade, leading to cell proliferation and survival. Several studies highlighted a critical role for MKK3 in tumor progression and invasion, and we previously identified MKK3 as transcriptional target of mutant (mut) p53 to sustain cell proliferation and survival, thus rendering MKK3 a promising target for anticancer therapies. Here, we found that targeting MKK3 with RNA interference, in both wild-type (wt) and mutp53-carrying cells, induced endoplasmic reticulum stress and autophagy that, respectively, contributed to stabilize wtp53 and degrade mutp53. MKK3 depletion reduced cancer cell proliferation and viability, whereas no significant effects were observed in normal cellular context. Noteworthy, MKK3 depletion in combination with chemotherapeutic agents increased tumor cell response to the drugs, in both wtp53 and mutp53 cancer cells, as demonstrated by enhanced poly (ADP-ribose) polymerase cleavage and reduced clonogenic ability in vitro. In addition, MKK3 depletion reduced tumor growth and improved biological response to chemotherapeutic in vivo. The overall results indicate MKK3 as a novel promising molecular target for the development of more efficient anticancer treatments in both wtp53- and mutp53-carrying tumors.
Assuntos
MAP Quinase Quinase 3/antagonistas & inibidores , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Animais , Autofagia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Estresse do Retículo Endoplasmático , Feminino , Humanos , MAP Quinase Quinase 3/metabolismo , Camundongos Nus , Proteínas Mutantes/metabolismo , Neoplasias/patologia , Estabilidade Proteica , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary cultures of prepubertal rat Sertoli cells secrete two major tissue inhibitors of metalloproteinases: TIMP-1 (M(r) 28K) and TIMP-2 (M(r) 21 K). FSH stimulated Sertoli cell TIMP-1 and TIMP-2 activity in a time- and dose-dependent manner and also stimulated TIMP-1 and TIMP-2 protein and messenger RNA levels. These effects were mimicked by the cAMP analog, 8-bromo-cAMP, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The protein kinase C activating phorbol ester phorbol myristate acetate (TPA) stimulated TIMP-1 but not TIMP-2 activity and messenger RNA levels. Cycloheximide and actinomycin-D inhibited basal TIMP-1 and TIMP-2 activity and inhibited the ability of FSH, 8-bromo-cAMP, and TPA to stimulate TIMP activity. The protein kinase A (PKA) inhibitor AMP Rp isomer did not affect basal TIMP-1 and TIMP-2 activity or TPA-stimulated TIMP-1 activity. However, the PKA inhibitor markedly reduced FSH and 3-isobutyl-1-methylxanthine stimulation of TIMP-1 and TIMP-2 activity. FSH, 8-bromo-cAMP, and TPA stimuli induced DNA binding complexes capable of binding to a TIMP-1 AP-1 site consensus sequence oligonucleotide. The AP-1 site binding complex(es) induced by all three treatments reacted with antibodies directed broadly against fos and jun protooncogene families and against the specific family members c-fos, junB, and junD but not c-jun proteins. Constitutive cAMP response element binding activity capable of binding an artificial cAMP response element binding site oligonucleotide was demonstrated in Sertoli cell nuclear extracts. This activity was minimally modulated by FSH, 8-bromo-cAMP, or TPA treatment. In summary, Sertoli cells secrete TIMP-1 and TIMP-2 that can be coordinately up-regulated by FSH through a cAMP, PKA-dependent pathway. a convergence of TPA, FSH, and cAMP mediated signals in prepubertal Sertoli cells may occur with the induction of specific AP-1 site binding complex(es) containing jun and fos proteins. Our data suggest that FSH stimulation of TIMP-2 expression may be regulated independently to that of TIMP-1. We propose that the ability of FSH to stimulate Sertoli cell TIMP activity suggests a central role for this hormone in the control of extracellular matrix turnover during testicular development at the level of metalloproteinase inhibition.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Glicoproteínas/antagonistas & inibidores , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Células de Sertoli/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Glicoproteínas/genética , Masculino , Metaloendopeptidases/antagonistas & inibidores , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Maturidade Sexual , Estimulação Química , Inibidor Tecidual de Metaloproteinase-2 , Inibidores Teciduais de MetaloproteinasesRESUMO
A comparable pattern of morphological neuronal differentiation was induced in the human neuroblastoma cell line SMS-KCNR by treatment with either retinoic acid (RA) or exogenous laminin (LM). LM expression and synthesis by SMS-KCNR was increased upon RA treatment which involved the cell bound, rather than the secreted protein. These data suggest an involvement of LM in the neuroblastoma differentiation process manifested both as an ability of LM to induce a morphological neuronal differentiation and as a selective control on LM metabolism during RA induced neuronal differentiation.
Assuntos
Laminina/biossíntese , Neuroblastoma/metabolismo , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Colágeno/biossíntese , Fibronectinas/biossíntese , Imunofluorescência , Humanos , Laminina/farmacologia , Microscopia de Contraste de Fase , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
It has recently been shown that tumor cells can retain the ability to undergo senescence, while the capacity of bypassing senescence has been associated with tumor progression. In this report, we showed that v-Ha-ras-mediated transformation of already immortal C2C12 myoblasts can be associated with senesence, in a low amount during in vitro passages and, to a higher extent, affer cellular stress (cell culture alkalinkation), or DNA damage (doxorubicin treatment). The capacity to undergo replicative senescence is associated with a strong increase of wt-p53 transcriptional activity and p21WAF1 up-regulation. These biochemical activities are down-modulated in the cells that evade the massive replicative senescence after stressing stimuli. Altogether, these findings show that active ras can cause senescence during the transformation of already immortal cells in associaton with p53/p21WAF1 pathway activation and support the hypothesis that p53/p21WAF1 functional activity is important in maintaining the integrity of the senescence pathway during cellular transformation.
Assuntos
Transformação Celular Neoplásica/genética , Senescência Celular/genética , Ciclinas/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes ras , Proteína Supressora de Tumor p53/fisiologia , Animais , Divisão Celular/genética , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Genes p53 , Camundongos , Músculos/citologia , Músculos/metabolismo , Músculos/fisiologia , Transdução de Sinais/genética , Estresse Fisiológico/genética , Estresse Fisiológico/patologia , Ativação Transcricional , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
A group of 10 rats underwent spinal trauma by epidural spinal compression according to Tator's procedure. After 4 months from injury cortical and spinal SEP were recorded, and the spinal injured tract was examined by electronic microscope. We studied morphologic and functional changes showed after some months behind the trauma. This report demonstrates and compares the varying sensitivity of cortical and spinal SEP for revealing functional spinal damage; ultrastructural investigations testify to the existence of reparative processes of the nervous tissue.
Assuntos
Potenciais Somatossensoriais Evocados , Traumatismos da Medula Espinal/fisiopatologia , Animais , Estimulação Elétrica , Feminino , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Tempo de Reação , Nervo Isquiático/fisiologia , Córtex Somatossensorial/fisiopatologia , Medula Espinal/fisiopatologia , Medula Espinal/ultraestrutura , Traumatismos da Medula Espinal/patologiaRESUMO
An intestinal epithelial cell line (IEC-17), undergoing a process of progressive morphological differentiation, was analysed for expression and synthesis of the extracellular matrix glycoproteins, fibronectin (FN) and laminin (LM). FN and LM cell surface expression was detected by immunoelectron microscopy, while intracytoplasmic accumulation was shown by immunofluorescence. 35S-methionine metabolic labelling was also performed to demonstrate FN and LM synthesis by IEC-17. We have compared two different maturation stages of the cell culture and have found that either early epithelial monolayer cells or later multistratified organoid structure cells expressed and produced large amounts of both proteins. These results indicate that FN and LM are constantly present during the process of IEC-17 organoid maturation: we can hypothesize that the two proteins act as mediators of cell to cell and cell to substrate adhesion interactions and, probably, have an active regulatory role in the process of intestinal epithelial cell differentiation.
Assuntos
Fibronectinas/análise , Mucosa Intestinal/metabolismo , Laminina/análise , Animais , Diferenciação Celular , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células Epiteliais , Epitélio/metabolismo , Fibronectinas/biossíntese , Imunofluorescência , Intestinos/citologia , Laminina/biossíntese , Microscopia Eletrônica , Testes de Precipitina , RatosRESUMO
Different collagen types (I, III, IV, V) were identified in breast carcinoma desmoplastic tissue by ultrastructural analysis and immunoelectronmicroscopy. Type V collagen is present as a 12 nm. fibril in the stroma, either adjacent to the basement membrane or concentrated around the thicker fibers. Myofibroblasts, fibroblasts and tumor cells can be its major producers. Its possible function as a bridge between different collagens can be utilized, with different finality, by the same cells that have produced it. Furthermore, type V collagen can be involved in tumor invasion of the stroma and in all directional movements of tumor cells, as already demonstrated for other extracellular matrix components.
Assuntos
Neoplasias da Mama/química , Neoplasias da Mama/ultraestrutura , Carcinoma Intraductal não Infiltrante/química , Carcinoma Intraductal não Infiltrante/ultraestrutura , Colágeno/análise , Fibrose , Humanos , Microscopia ImunoeletrônicaRESUMO
TP53, one of the most important oncosuppressors, is frequently mutated in cancer. Several p53 mutant proteins escape proteolytic degradation and are highly expressed in an aberrant conformation often acquiring pro-oncogenic activities that promote tumor progression and resistance to therapy. Therefore, it has been vastly proposed that reactivation of wild-type (wt) function(s) from mutant p53 (mutp53) may have therapeutic significance. We have previously reported that Zn(II) restores a folded conformation from mutp53 misfolding, rescuing wild-type (wt) p53/DNA-binding and transcription activities. However, whether Zn(II) affects mutp53 stability has never been investigated. Here we show that a novel Zn(II) compound induced mutp53 (R175H) protein degradation through autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins. Accordingly, pharmacological or genetic inhibition of autophagy prevented Zn(II)-mediated mutp53H175 degradation as well as the ability of the Zn(II) compound to restore wtp53 DNA-binding and transcription activity from this mutant. By contrast, inhibition of the proteasome failed to do so, suggesting that autophagy is the main route for p53H175 degradation. Mechanistically, Zn(II) restored the wtp53 ability to induce the expression of the p53 target gene DRAM (damage-regulated autophagy modulator), a key regulator of autophagy, leading to autophagic induction. Accordingly, inhibition of wtp53 transactivation by pifithrin-α (PFT-α) impaired both autophagy and mutp53H175 degradation induced by curcumin-based zinc compound (Zn(II)-curc). Viewed together, our results uncover a novel mechanism employed by Zn(II)-curc to reactivate mutp53H175, which involves, at least in part, induction of mutp53 degradation via wtp53-mediated autophagy.
Assuntos
Autofagia/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Compostos de Zinco/farmacologia , Linhagem Celular Tumoral , Curcumina/química , Células HCT116 , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Compostos de Zinco/químicaAssuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Caspase 8/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , FosforilaçãoRESUMO
Tumor cell tolerance to nutrient deprivation can be an important factor for tumor progression, and may depend on deregulation of both oncogenes and oncosuppressor proteins. Homeodomain-interacting protein kinase 2 (HIPK2) is an oncosuppressor that, following its activation by several cellular stress, induces cancer cell death via p53-dependent or -independent pathways. Here, we used genetically matched human RKO colon cancer cells harboring wt-HIPK2 (HIPK2(+/+)) or stable HIPK2 siRNA interference (siHIPK2) to investigate in vitro whether HIPK2 influenced cell death in glucose restriction. We found that glucose starvation induced cell death, mainly due to c-Jun NH2-terminal kinase activation, in HIPK2(+/+)cells compared with siHIPK2 cells that did not die. (1)H-nuclear magnetic resonance quantitative metabolic analyses showed a marked glycolytic activation in siHIPK2 cells. However, treatment with glycolysis inhibitor 2-deoxy-D-glucose induced cell death only in HIPK2(+/+) cells but not in siHIPK2 cells. Similarly, siGlut-1 interference did not re-establish siHIPK2 cell death under glucose restriction, whereas marked cell death was reached only after zinc supplementation, a condition known to reactivate misfolded p53 and inhibit the pseudohypoxic phenotype in this setting. Further siHIPK2 cell death was reached with zinc in combination with autophagy inhibitor. We propose that the metabolic changes acquired by cells after HIPK2 silencing may contribute to induce resistance to cell death in glucose restriction condition, and therefore be directly relevant for tumor progression. Moreover, elimination of such a tolerance might serve as a new strategy for cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Desoxiglucose/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Desoxiglucose/uso terapêutico , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaboloma , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Zinco/farmacologiaRESUMO
Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-µ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.
Assuntos
Antineoplásicos/farmacologia , Autofagia , Catepsina D/metabolismo , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Lisossomos/efeitos dos fármacos , Sulfonamidas/farmacologia , Transporte Ativo do Núcleo Celular , Fator de Indução de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Catepsina D/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Linfoma de Efusão Primária , Lisossomos/enzimologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Pepstatinas/farmacologia , Permeabilidade , Inibidores de Proteases/farmacologiaRESUMO
The p53 protein is the most studied tumor suppressor and the p53 pathway has been shown to mediate cellular stress responses that are disrupted when cancer develops. After DNA damage, p53 is activated as transcription factor to directly induce the expression of target genes involved in cell-cycle arrest, DNA repair, senescence and, importantly, apoptosis. Post-translational modifications of p53 are essential for the activation of p53 and for selection of target genes. The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating its N-terminal serine 46 (Ser46) and facilitating Lys382 acetylation at the C-terminus. HIPK2 is activated by numerous genotoxic agents and can be deregulated in tumors by several conditions including hypoxia. Recent findings suggest that HIPK2 active/inactive protein can affect p53 function in multiple and unexpected ways. This makes p53 as well as HIPK2 interesting targets for cancer therapy. Hence, understanding the role of HIPK2 as p53 activator may provide important insights in the process of tumor progression, and may also serve as the crucial point in the diagnostic and therapeutical aspects of cancer.
Assuntos
Proteínas de Transporte/fisiologia , Neoplasias/terapia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Immunoelectron microscopy was employed to detect laminin and fibronectin cell surface expression on five Ewing's sarcoma lines plus a normal fibroblast line as control. Monospecific antibodies to both glycoproteins were detected on tumour cell and fibroblast layers with colloidal gold--protein A conjugates. All five tumour lines were positive for fibronectin and/or laminin, whereas the fibroblast line expressed fibronectin only, as expected. Fibronectin displayed a dense granular pattern, typically in the cell-cell and cell-matrix adhesion areas; laminin displayed a punctate pattern. 3H-leucine metabolical labelling was also used to demonstrate laminin and fibronectin synthesis. The labelled proteins released in the culture media were separated by molecular weight on SDS-PAGE and identified by immunoprecipitation with the monospecific antibodies. The results substantiated the immunoelectron microscopy data. These findings indicate that Ewing's sarcoma lines produce a complex extracellular matrix including fibronectin and laminin, in addition to the collagens described by other workers. Histogenetic classification of this tumour in terms of extracellular matrix proteins synthesis is thus more difficult than has been supposed. The same complexity must also be borne in mind when using the matrix components as an aid to Ewing's sarcoma differentiation from other childhood tumours.
Assuntos
Fibronectinas/biossíntese , Laminina/biossíntese , Sarcoma de Ewing/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Técnicas Imunológicas , Microscopia Eletrônica , Sarcoma de Ewing/ultraestruturaRESUMO
BACKGROUND: Expression of exogenous wild-type p53 (wt-p53) protein in tumor cells can suppress the transformed phenotype whereas it does not apparently induce detrimental effects in non-transformed cells. This observation may provide a molecular basis for p53-mediated gene therapy of p53-sensitive cancers without the need for tumor targeting. METHODS: To understand the molecular mechanisms responsible for this different behavior in tumor versus normal cells, biochemical and functional analyses of exogenous wt-p53 protein were performed on non-transformed C2C12 myoblasts and their transformed counterparts, the C2-ras cells. RESULTS: The exogenous wt-p53 protein, which induced persistent growth arrest only in transformed C2-ras cells, was shown to be significantly more stable in transformed than in non-transformed cells. This different stability was due to different p53 proteolytic degradation. Moreover, constitutively, exogenous wt-p53 protein was found to be transcriptionally active only in C2-ras cells but it could also be activated in C2C12 cells by genotoxic damage. CONCLUSIONS: Non-transformed C2C12 cells present regulatory system(s) which control the expression and the activity of exogenously expressed wt-p53 protein probably through degradation and maintenance in a latent form. This regulatory system is lost/inactivated upon transformation.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares , Proteína Supressora de Tumor p53/genética , Adenoviridae/genética , Animais , Western Blotting , Divisão Celular , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cisteína Endopeptidases/farmacologia , Dano ao DNA/fisiologia , DNA Recombinante/genética , Doxorrubicina/farmacologia , Técnicas de Transferência de Genes , Genes Reporter/genética , Genes ras/genética , Vetores Genéticos/genética , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Complexos Multienzimáticos/farmacologia , Músculos/citologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The ultrastructural pattern of the anti-tumor response elicited by interleukin-4 (IL-4) was investigated by using a spontaneous mammary adenocarcinoma (TS/A) unable to elicit protective immunity in syngeneic BALB/c mice as suggested by a variety of preimmunization-challenge experiments. A subcutaneous lethal challenge of TS/A tumor cells was inhibited in a significant number of BALB/c mice receiving recombinant murine IL-4 injected daily for 10 days around the tumor-draining lymph node. Tumor rejection was mainly the result of direct membrane and cytoplasmic damage to tumor cells by eosinophils, neutrophils and macrophages that deeply penetrated the proliferating tumor mass. Lymphocytes and fibroblasts participated in the reaction by interacting with tumor cells, granulocytes and each other. The most frequent cell interactions in the peri- and intra-tumoral areas and in the tumor-draining lymph nodes are illustrated. The efficiency with which the IL-4-activated reaction leads to tumor inhibition and induction of a T-lymphocyte-dependent tumor-specific immune memory appears to depend on interactions between distinct leukocytes.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/ultraestrutura , Interleucina-4/farmacologia , Linfócitos do Interstício Tumoral/fisiologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/ultraestrutura , Adenocarcinoma/patologia , Animais , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Matriz Extracelular/fisiologia , Feminino , Injeções Intralinfáticas , Soluções Isotônicas/farmacologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/fisiologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Necrose , Proteínas Recombinantes/farmacologiaRESUMO
Athymic nude (nu/nu) mice are widely employed for the heterotransplantation of human tumor cell lines established in vitro and tumor cells directly grafted from patients. By contrast, hemopoietic malignancies have consistently proved difficult to transplant and well-characterized human leukemias suitable for studies in nude mice are scarce. We report here our experience with subcutaneous xenotransplantation of human neoplastic cells into nu/nu mice immunosuppressed through sublethal irradiation and splenectomy (SI-nu/nu) and with an additional injection of anti-Asialo-GM1 antibodies (SIA-nu/nu) in order to eliminate natural killer activity. Thirteen out of 16 continuous cell lines established in vitro from solid tumors and 7 out of 14 human tumors obtained from fragments of surgical specimens formed a progressively growing tumor in SI-nu/nu mice. Six out of 8 in vitro established human leukemic cell lines and 5 out of 18 neoplastic hematopoietic cells directly xenotransplanted from the patient grew SIA-nu/nu mice. When the membrane and chromosome markers of neoplastic cells that grew into the mice were evaluated, only marginal differences with those of the original tumors were found. In addition, when interfering factors alter the histological aspect of the primary tumor, xenotransplantation may also be of some help in histological diagnosis. By using SI- and SIA-nu/nu mice, it is thus possible to build up several new in vivo experimental systems with fresh human tumors that may be of value in studying the efficacy of differentiation factors and immunological maneuvers on the in vivo growth of human tumors.
Assuntos
Leucemia Experimental/patologia , Linfoma/patologia , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Animais , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante Heterólogo , Células Tumorais Cultivadas/transplanteRESUMO
The ability of NK cells to synthesize and secrete fibronectin (FN), an extracellular matrix glycoprotein which plays a key role in many biologic processes including cellular adhesion, morphology, cytoskeletal organization, cell migration, and invasiveness, was studied. By using affinity-purified polyclonal antibodies directed against human cellular or plasma FN, the presence of FN was evidentiated on Percoll-purified rat large granular lymphocyte or on a large granular lymphocyte tumor cell line (CRC) by flow cytometry and immunoelectron microscopy. Its expression increased after NK cell activation by poly I:C administration. Biochemical analysis by immunoprecipitation and SDS-PAGE indicated that FN was associated to cell surface and secreted in the supernatant in a molecular form similar to that of FN from L929 fibroblasts. In an attempt to understand the role of FN in the NK cell function, we found that an antibody against human plasma FN and its F(ab')2 fragment inhibited NK cytotoxicity against YAC-1 target at the effector cell level. Inhibition occurred at the postbinding level, because F(ab')2 anti-FN inhibited induction of phosphatidylinositol hydrolysis by YAC-1 target cells, whereas binding to target cells was not affected. The possible role of FN in the NK cytotoxic function is suggested.