RESUMO
OBJECTIVE: In this study, we aimed to clarify the precise mechanism underlying lipopolysaccharide (LPS)-induced osteoclastogenesis in periodontal disease with a special reference to double-stranded RNA-dependent protein kinase (PKR). MATERIAL AND METHODS: We dissected the role of PKR in LPS-induced osteoclast differentiation and function using primary mouse bone marrow cells and RAW264.7 pre-osteoclastic cell line. We used a rat experimental periodontitis (PD) model induced by ligature placement with a Porphyromonas gingivalis LPS injection (PD rat) and analyzed the therapeutic effects of C16, a PKR inhibitor, on bone loss in PD rats. RESULTS: Protein kinase is strongly upregulated and phosphorylated by LPS in the osteoclasts. The inhibition of PKR suppressed LPS-stimulated osteoclast formation and activation. PKR inhibition also suppressed the LPS-mediated activation of NF-κB and MAPK, which are critical pathways for osteoclastogenesis. High expressions of PKR were detected in osteoclasts of PD rats, and the treatment with C16 effectively prevented alveolar bone destruction in PD rats. CONCLUSIONS: PKR plays a pivotal role in LPS-induced bone loss in PD and, thus, has potential as a therapeutic target for PD.
Assuntos
Indóis/uso terapêutico , Osteogênese/efeitos dos fármacos , Doenças Periodontais/tratamento farmacológico , Doenças Periodontais/metabolismo , Tiazóis/uso terapêutico , eIF-2 Quinase/metabolismo , Perda do Osso Alveolar/prevenção & controle , Animais , Linhagem Celular , Indóis/farmacologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Ratos , Tiazóis/farmacologia , eIF-2 Quinase/antagonistas & inibidoresRESUMO
The mak gene encodes a new protein kinase distantly related to cdc2 kinase, and its transcripts are expressed exclusively in testicular germ cells at and after meiosis (H. Matsushime, A. Jinno, N. Takagi, and M. Shibuya, Mol. Cell. Biol. 10:2261-2268, 1990). In this study, we prepared a series of antibodies against synthetic peptides and fusion products of the mak gene and characterized the subcellular localization, protein kinase activity, and association with other cellular proteins of Mak. Mak products were identified as 66- and 60-kDa proteins that specifically appeared in rat testes after puberty. Testicular germ cell fractionation revealed that Mak products were most abundant in the fraction of the late pachytene stage and that their levels were dramatically decreased in postmeiotic haploid cells. Mak products were localized mostly in the cytoplasm as a soluble form. [35S]methionine labelling demonstrated that Mak products were associated with a 210-kDa cellular protein; in an in vitro kinase assay with immunoprecipitates of Mak products, the 210-kDa cellular protein was efficiently phosphorylated on serine and threonine residues. Furthermore, in a testicular cell culture system with 32Pi, the 210-kDa molecule associated with Mak was phosphorylated in vivo on serine and threonine residues. These results strongly suggest that the Mak complex may play a role in meiosis during spermatogenesis and that a phosphorylated 210-kDa protein is one of the physiological substrates for this protein kinase.
Assuntos
Fosfoproteínas/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Espermatogênese , Testículo/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Gel , Masculino , Meiose , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Maturidade Sexual , Testículo/crescimento & desenvolvimentoRESUMO
To study the mechanism of vitamin A and FSH action on testicular germ cell differentiation, artificially induced cryptorchid testes of adult mice were cultured in vitro, because such testes contain only type A spermatogonia and Sertoli cells in their seminiferous tubules. A synergistic effect of vitamin A compounds with FSH, but not with LH, on testicular germ cell differentiation was observed. Our data suggested that FSH stimulated the proliferation of type A spermatogonia, whereas retinoids induce spermatogenesis from type A spermatogonia into intermediate or type B spermatogonia. Possible mechanisms of action of retinoids and FSH on testicular germ cell differentiation are discussed.
Assuntos
Criptorquidismo/fisiopatologia , Hormônio Foliculoestimulante/farmacologia , Espermatogônias/fisiologia , Espermatozoides/fisiologia , Testículo/fisiopatologia , Vitamina A/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testículo/efeitos dos fármacosRESUMO
Surgically prepared cryptorchid mouse testes containing only type A spermatogonia were cultured with (Bu)2cAMP in combination with vitamin A (retinol). Treatment with (Bu)2cAMP and retinol for 12-24 h and with basal medium for an additional 8 days stimulated mitotic activity in type A spermatogonia and induced differentiation of germ cells. However, (Bu)2cAMP alone did not induce differentiation of type A spermatogonia. Moreover, when cryptorchid testes were treated with (Bu)2cAMP for longer than 3 days in the presence or absence of retinol, differentiation of type A spermatogonia did not take place; disintegration of the seminiferous tubules occurred instead. When the cryptorchid testes were cultured for 24 h in a medium containing a fixed concentration of retinol and varying concentrations of (Bu)2cAMP from 0.001-0.4 mM, there was a dose-dependent increase in the number of differentiated and mitotic germ cells and type A spermatogonia. Likewise, at a fixed dose of (Bu)2cAMP and increasing concentrations of retinol, a dose-dependent increase in the number of differentiated and mitotic germ cells occurred. However, the number of type A spermatogonia was decreased. The addition of puromycin, cycloheximide, and actinomycin D to the medium completely blocked retinol-(Bu)2cAMP-induced differentiation of the germ cells. The present results suggest that cAMP and retinol trigger biochemical events promoting the synthesis of specific macromolecules involved in the proliferation and differentiation of type A spermatogonia.
Assuntos
Bucladesina/farmacologia , Criptorquidismo/patologia , Espermatogônias/citologia , Espermatozoides/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Germinativas/citologia , Técnicas In Vitro , Masculino , Camundongos , Espermatogênese/efeitos dos fármacos , Vitamina A/farmacologiaRESUMO
We isolated a rat cDNA encoding part of the beta-isotype of the B regulatory subunit (BR beta) of protein phosphatase 2A (PP2A). The isolated cDNA encoded the region corresponding to amino acids positions 8(R) to 177(N) of human BR beta. The identities of the nucleotide and amino acid sequences of the rat and human BR beta s were 95.7% and 100%, respectively. The BR beta mRNA was specifically expressed in rat brain and testis, the lengths of mRNAs in these two organs being different. In the testis, the BR beta mRNA was first detected 40 days after birth, increasing gradually thereafter, and was expressed specifically in elongated spermatids, while mRNA of the alpha-isotype (BR alpha) was expressed equally in all spermatogenic cells. After meiosis, round spermatids change morphologically to elongated spermatids. BR beta may regulate the activity of the PP2A catalytic subunit in spermatids, and be involved in spermatogenic maturation, especially spermatid elongation.
Assuntos
Isoenzimas/genética , Fosfoproteínas Fosfatases/genética , Espermátides/enzimologia , Espermatócitos/enzimologia , Testículo/enzimologia , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Expressão Gênica , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Proteína Fosfatase 2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Valores de Referência , Testículo/crescimento & desenvolvimentoRESUMO
We have analyzed the distribution of the gamma 1 isotype of rat protein phosphatase type 1 catalytic subunit in rat salivary glands. Formaldehyde-fixed paraffin sections were reacted with the PP1 gamma 1 antibody using an immunohistochemical method. Positive staining occurred in striated ducts of parotid gland. However, the staining reaction was less intense in submandibular gland. Proteins were also prepared from rat salivary glands and subjected to SDS-PAGE, followed by Western blotting analysis with the PP1 gamma 1 antibody. The antibody interacted with protein corresponding to an estimated molecular mass of 36 kDa present in the parotid gland. The staining reaction was considerably weaker with the proteins from submandibular gland.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Glândulas Salivares/enzimologia , Animais , Western Blotting , Técnicas Imunoenzimáticas , Masculino , Glândula Parótida/enzimologia , Proteína Fosfatase 1 , Ratos , Ratos WistarRESUMO
In order to study hormonal effects on testicular germ cell differentiation, especially on type A spermatogonia, artificially induced cryptorchid testes of adult mice were cultured in a medium containing testosterone, dihydrotestosterone, tri-iodothyronine, dibutyryl 3':5' cyclic adenosine monophosphate, human chorionic gonadotrophin, LH, FSH, insulin and transferrin. These substances, with the exception of FSH, showed no stimulatory effect on the differentiation of type A spermatogonia. However, FSH activated cell division in type A spermatogonia and stimulated them to differentiate, while LH showed neither the promotion of differentiation nor a synergistic effect on FSH-mediated germ cell differentiation.
Assuntos
Criptorquidismo/metabolismo , Hormônios/farmacologia , Espermatogônias/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Androgênios/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Espermatogônias/efeitos dos fármacosRESUMO
The effect of dibutyryl cyclic AMP (dbcAMP) on DNA synthesis in mouse cryptorchid explants with only type A spermatogonia was examined in vitro. Low concentration of dbcAMP (0.08 mmol/l) stimulated DNA synthesis by germ cells but inhibited that by non-germ cells.
Assuntos
Bucladesina/farmacologia , DNA/biossíntese , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Autorradiografia , Criptorquidismo/metabolismo , Técnicas de Cultura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatozoides/metabolismo , Testículo/metabolismo , Teofilina/farmacologiaRESUMO
The effect of epidermal growth factor (EGF) on testicular germ cell differentiation was investigated. Testicular fragments from surgically prepared cryptorchid testes of adult mice were cultured for 9 days in serum-free media containing various concentrations of EGF. Histological sections of testis were examined under a light microscope and each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. EGF at concentrations ranging from 100 to 200 ng/ml induced differentiation of type A spermatogonia. The observed maximal stimulatory activity of EGF at a concentration of 100 ng/ml was 30% of the positive control cultures treated with calf serum. EGF at concentrations ranging from 1 to 100 ng/ml significantly inhibited the mitotic activity of FSH, FSH plus retinol, or FSH plus fetuin on type A spermatogonia and their differentiation. The number of type A spermatogonia in testes cultured with FSH, FSH plus retinol, or FSH plus fetuin decreased when EGF was added. On the other hand, EGF stimulated the differentiation of type A spermatogonia induced with fetuin but did not influence retinol-induced differentiation. It is proposed that EGF inhibits testicular germ cell differentiation by blocking the proliferation of type A spermatogonia stimulated by FSH.
Assuntos
Criptorquidismo/patologia , Fator de Crescimento Epidérmico/farmacologia , Espermatogônias/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Hormônio Foliculoestimulante/farmacologia , Inibidores do Crescimento/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitose/efeitos dos fármacos , Espermatogônias/patologia , Vitamina A/farmacologia , alfa-Fetoproteínas/farmacologiaRESUMO
In order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3]. A physiological concentration of 1 alpha, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites--1 alpha, 24-dihydroxyvitamin D3 [1 alpha, 24 (OH)2D3], 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3]--also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1,000 times greater than that of 1 alpha, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.
Assuntos
Fosfatase Alcalina/genética , Calcitriol/farmacologia , Colecalciferol/farmacologia , Osteoblastos/enzimologia , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Osso e Ossos/enzimologia , Linhagem Celular , Células Clonais , Indução Enzimática , Cinética , Camundongos , Osteoblastos/efeitos dos fármacosRESUMO
The kinetics of DNA synthesis of whole testis in mice was studied in artificial unilateral cryptorchidism and in its surgical reversal. Cryptorchidism impaired total testicular DNA synthesis. Approximately 1 month after the operation, activity of testicular DNA synthesis decreased to about 30% that of the contralateral intact testis. A complete recovery of DNA synthesis was observed within 1 month after the surgical reversal of cryptorchidism.
Assuntos
Criptorquidismo/cirurgia , DNA/biossíntese , Testículo/análise , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Testículo/anatomia & histologia , Testículo/fisiologia , Uridina/metabolismoRESUMO
The Fas antigen is a cell-surface glycoprotein that mediates apoptosis from the cell surface into the cytoplasm. Polyclonal antibody (Fas D) was raised against a synthetic polypeptide selected from the extracellular part of the human Fas antigen (amino acid residues 104-114) and was used to detect the Fas antigen in human gingiva. Biopsy specimens of human gingiva were prepared, and the paraffin sections were reacted with the Fas D antibody by an immunohistochemical method. The antibody localized to the prickle-cell layer and to granular layer keratinocytes of human gingiva. Proteins were also prepared from human gingiva and subjected to SDS-PAGE, followed by Western-blotting analysis with the Fas D antibody. The antibody interacted with a band corresponding to an estimated molecular weight of 35 kDa. The incidence of the immunoreactive 35-kDa protein was detected in the gingiva of 90% of the 20 individuals examined. The Fas antigen detected in human gingiva may be related to the physiological turnover of oral mucosa.
Assuntos
Gengiva/imunologia , Mucosa Bucal/imunologia , Receptor fas/análise , Sequência de Aminoácidos , Apoptose , Western Blotting , Eletroforese em Gel de Poliacrilamida , Gengiva/citologia , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Peso MolecularRESUMO
Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 microg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 microg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 microg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.
Assuntos
Aggregatibacter actinomycetemcomitans/química , Apoptose , Proteínas de Bactérias/farmacologia , Osteoblastos/microbiologia , Osteoblastos/fisiologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citotoxinas/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Prevotella intermedia/química , Sonicação , Células Tumorais CultivadasRESUMO
Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to Fas receptor leads to activation of the latter and the induction of intracellular signals that result in apoptotic cell death. In the present study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis to examine the expression of mRNAs and proteins of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. The PCR product of Fas receptor mRNA was detected in the cells and a protein with an estimated molecular weight of 35,000 was also expressed in them. Expression of Fas receptor mRNA stimulated by okadaic acid was elevated in dose- and time-dependent manners as judged by semiquantitative RT-PCR analysis, with the maximum expression level at 50 nM and 8 h treatment. Fas ligand mRNA expression was also stimulated by okadaic acid in SCC-25 cells in dose- and time-dependent manners. Okadaic acid also stimulated the expression of Fas ligand protein in the cells. Okadaic acid in serum-free medium induced apoptosis in SCC-25 cells in a time-dependent manner up to 24 h as determined by nuclear condensation and fragmentation of chromatin and DNA ladder formation. The present results indicate that the expression of Fas receptor and Fas ligand is negatively regulated by a protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in SCC-25 cells. Our results also suggest that Fas receptor and Fas ligand system might regulate apoptosis in SCC-25 cells in an autocrine fashion.
Assuntos
Apoptose/fisiologia , Carcinoma de Células Escamosas/patologia , Inibidores Enzimáticos/farmacologia , Glicoproteínas de Membrana/análise , Proteínas de Neoplasias/análise , Ácido Okadáico/farmacologia , Neoplasias da Língua/patologia , Receptor fas/análise , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma de Células Escamosas/química , Meios de Cultura Livres de Soro , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida/métodos , Proteína Ligante Fas , Humanos , Glicoproteínas de Membrana/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Língua/química , Células Tumorais Cultivadas/patologia , Receptor fas/efeitos dos fármacosRESUMO
Oral squamous carcinoma cell line SSCKN cells were shown to be highly sensitive to bleomycin, whereas SCCTF cells were minimally sensitive to this reagent. To determine whether the anticancer drug resistance to oral squamous carcinoma cells could be related to the degree of the drug-induced apoptosis, we examined the effects of peplomycin on induction of apoptosis in these cells. After reaching subconfluence, SCCKN and SCCTF cells were exposed to various concentrations of peplomycin. Peplomycin caused cytotoxicity in both SCCKN and SCCTF cells in a dose-dependent fashion with the maximal effect at concentrations of 1 and 10 microM, respectively, as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, we observed marked nuclear condensation and fragmentation of chromatin in SCCKN cells treated with 1 microM peplomycin. However, SCCTF cells treated with 1 microM peplomycin showed neither nuclear condensation nor fragmentation. DNA ladder formation was also detected in both cell lines by treatment with peplomycin. The induced DNA ladder formation in SCCKN and SCCTF cells was dose-dependent, with the maximal effect at concentrations of 5 and 50 microM, respectively. Bleomycin also induced DNA ladder formation in SCCKN and SCCTF cells with different sensitivities. Mitomycin C induced DNA laddering in both SCCKN and SCCTF cells; however, the intensity of DNA ladder formation was almost the same in both cell lines. The present results indicate that peplomycin-induced apoptosis in oral squamous carcinoma cell lines depends on the sensitivity of these cells to bleomycin.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Bleomicina/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Peplomicina/uso terapêutico , Carcinoma de Células Escamosas/patologia , Colorimetria , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Humanos , Microscopia de Contraste de Fase , Mitomicina/uso terapêutico , Neoplasias Bucais/patologia , Resultado do Tratamento , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in oral epithelial cells we examined the effects of protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CA), on cultured human oral squamous carcinoma (SCC) cell line, SCC-25 cells. After reaching subconfluence these cells were exposed to varying concentrations of the protein phosphatase inhibitors, OA and CA. Both OA and CA induced cell death in SCC-25 cells in a dose-dependent fashion as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin was observed. DNA ladder formation also was detected in SCC-25 cells by treatment with OA and CA. The induced nuclear fragmentation and DNA ladder formation were dose-dependent with maximal effect at concentrations of 20 nM OA and 2 nM CA, respectively. OA also induced DNA ladder formation in other human oral SCC cell lines, SCCKN and SCCTF. To further determine if new gene transcription and protein synthesis are required for OA-induced apoptosis in SCC-25 cells, the cells were treated for 48 h with varying concentrations of cycloheximide in the presence of 20 nM OA. Cycloheximide did not protect the cells against OA-induced cytotoxicity and DNA ladder formation. Based on the known selectivity of OA and CA, the present results indicate that the pathway of the apoptosis in the cultured oral SCC cells is in part regulated by protein phosphatase type 1 and type 2A. Our results also indicate that new protein synthesis is not involved in OA-induced apoptosis in SCC-25 cells.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Neoplasias Bucais/tratamento farmacológico , Ácido Okadáico/uso terapêutico , Oxazóis/uso terapêutico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Humanos , Toxinas Marinhas , Neoplasias Bucais/patologia , Células Tumorais CultivadasRESUMO
We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level.
Assuntos
Carcinógenos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces , Neoplasias Bucais/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Ácido Okadáico/uso terapêutico , Fatores de Transcrição/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce , Eletroforese em Gel de Ágar/métodos , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Supressão Genética , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Glicoproteínas de Membrana/efeitos dos fármacos , Ácido Okadáico/farmacologia , Neoplasias da Língua/metabolismo , Receptor fas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteína Ligante Fas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/metabolismo , Ligantes , Glicoproteínas de Membrana/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.
Assuntos
Antígenos de Superfície/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Ductos Salivares/efeitos dos fármacos , Glândula Submandibular/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Receptor fas/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Pareamento de Bases , Linhagem Celular , Relação Dose-Resposta a Droga , Proteína Ligante Fas , Regulação da Expressão Gênica , Humanos , Immunoblotting , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Peso Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Paraffin sections of biopsy specimens obtained from 46 patients with oral squamous cell carcinomas were stained with both anti-peptide antibody against human Fas antigen and monoclonal mouse antibody against human proliferating cell nuclear antigen (PCNA). The patients received chemotherapy with a combination of carboplatin and peplomycin sulfate or mitomycin C and peplomycin sulfate before surgery. The relation between the expression of Fas antigen and the clinical features of each case was examined. The correlation between PCNA and Fas antigen expression was also studied. The mean PCNA labeling index of the 22 Fas-negative cases was 46.9%, which was significantly higher than that of the 24 Fas-positive cases (39.5%). Strong correlations were found between the expression of Fas antigen and the response to chemotherapy, tumor recurrence, and survival. The Fas-negative group had only a minor response to chemotherapy and a poor outcome, whereas the Fas-positive group had a better response to chemotherapy and a good outcome. Although lymph node metastasis was significantly related to survival, there was no correlation between Fas antigen expression and lymph node metastasis. The Kaplan-Meier survival curve of patients positive for Fas antigen was significantly better than that of patients negative for Fas antigen. Our results suggest that Fas antigen expression is an independent predictor of outcome whose usefulness should be evaluated in prospective studies.