RESUMO
The integrin αvß6 activates latent transforming growth factor-ß (TGF-ß) within the kidney and may be a target for the prevention of chronic allograft fibrosis after kidney transplantation. However, TGF-ß also has known immunosuppressive properties that are exploited by calcineurin inhibitors (CNIs); thus, the net benefit of αvß6 inhibition remains undetermined. To assess the acute impact of interference with αvß6 on acute rejection, we tested a humanized αvß6-specific monoclonal antibody (STX-100) in a randomized, double-blinded, placebo-controlled nonhuman primate renal transplantation study to evaluate whether αvß6 blockade alters the risk of acute rejection during CNI-based immunosuppression. Rhesus monkeys underwent renal allotransplantation under standard CNI-based maintenance immunosuppression; 10 biopsy-confirmed rejection-free animals were randomized to receive weekly STX-100 or placebo. Animals treated with STX-100 experienced significantly decreased rejection-free survival compared to placebo animals (p = 0.049). Immunohistochemical analysis confirmed αvß6 ligand presence, and αvß6 staining intensity was lower in STX-100-treated animals (p = 0.055), indicating an apparent blockade effect of STX-100. LAP, LTBP-1 and TGF-ß were all decreased in animals that rejected on STX-100 compared to those that rejected on standard immunosuppression alone, suggesting a relevant effect of αvß6 blockade on local TGF-ß. These data caution against the use of αvß6 blockade to achieve TGF-ß inhibition in kidney transplantation.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Imunossupressores/efeitos adversos , Integrinas/antagonistas & inibidores , Transplante de Rim/métodos , Aloenxertos , Animais , Anticorpos Monoclonais/química , Antígenos de Neoplasias , Biópsia , Rejeição de Enxerto , Terapia de Imunossupressão , Macaca mulatta , Projetos Piloto , Distribuição Aleatória , Fator de Crescimento Transformador beta1/sangueRESUMO
BACKGROUND: Current organotypic models of dysplasia and oral squamous cell carcinoma (OSCC) lack the complexity that mimics in vivo tissue. Here we describe a three-dimensional in vitro model of the oral epithelium that replicates tumour progression from dysplasia to an invasive phenotype. METHODS: The OSCC cell lines were seeded as a cell suspension (D20, Cal27) or as multicellular tumour spheroids (FaDu) with oral fibroblasts on to a de-epidermised acellular dermis to generate tissue-engineered models and compared with patient biopsies. RESULTS: The D20 and Cal27 cells generated a model of epithelial dysplasia. Overtime Cal27 cells traversed the basement membrane and invaded the connective tissue to reproduce features of early invasive OSCC. When seeded onto a model of the normal oral mucosa, FaDu spheroids produced a histological picture mimicking carcinoma in situ with severe cellular atypia juxtaposed to normal epithelium. CONCLUSION: It is possible to culture in vitro models with the morphological appearance and histological characteristics of dysplasia and tumour cell invasion seen in vivo using native dermis. Such models could facilitate study of the molecular processes involved in malignant transformation, invasion and tumour growth as well as in vitro testing of new treatments, diagnostic tests and drug delivery systems for OSCC.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/patologia , Engenharia Tecidual , Citometria de Fluxo , Humanos , Imuno-HistoquímicaRESUMO
Cervical squamous cell carcinomas are composed histologically of tumour cell islands surrounded by varying amounts of tumour stroma, the amount and composition of which are influenced by local TGF-beta(1). TGF-beta(1) is secreted in an inactive complex with latency-associated peptide (LAP). Both LAP and the extracellular matrix (ECM) protein fibronectin are important ligands for the integrin receptor alpha v beta 6. While alpha v beta 6 is only weakly expressed by normal epithelia, it is up-regulated in different carcinomas where it generally reflects a more aggressive phenotype. In cervical cancer, the expression of alpha v beta 6 has not thus far been investigated. Given the ability of alpha v beta 6 both to activate TGF-beta(1) and to interact with fibronectin, we studied correlations between the expression of these components and disease parameters in a large cohort of cervical cancer specimens. We analysed alpha v beta 6 expression using immunohistochemistry in primary cervical squamous carcinomas of FIGO stage IA to IIB patients and correlated the findings with formerly investigated fibronectin and TGF-beta(1) expression and clinico-pathological parameters. alpha v beta 6 expression was also examined in cervical intra-epithelial neoplasia (CIN) and lymph node metastases. alpha v beta 6 was only weakly expressed in normal epithelium but clearly up-regulated in CIN lesions. In carcinomas, strong expression of alpha v beta 6 in tumour cells correlated with different clinico-pathological parameters and with worse overall and disease-free survival. Furthermore, alpha v beta 6 expression correlated positively with TGF-beta(1) mRNA expression as well as with fibronectin expression. Overexpression of alpha v beta 6 in cervical squamous carcinomas is an unfavourable prognostic factor. This might reflect an increased capacity of alpha v beta 6-expressing tumour cells to migrate in a fibronectin-rich ECM and/or to activate TGF-beta(1) at the tumour/stroma interface, both of which processes may contribute to cervical cancer progression.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Integrinas/genética , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Carcinoma in Situ/química , Carcinoma in Situ/mortalidade , Carcinoma in Situ/patologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Colo do Útero/química , Colo do Útero/patologia , Progressão da Doença , Feminino , Fibronectinas/análise , Humanos , Imuno-Histoquímica , Hibridização In Situ/métodos , Integrinas/análise , Metástase Linfática , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/análise , Taxa de Sobrevida , Fator de Crescimento Transformador beta/genética , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaAssuntos
Benzamidas/síntese química , Osso e Ossos/enzimologia , Ácido Cítrico/química , Cicloexanos/síntese química , Inibidores Enzimáticos/síntese química , Compostos Organofosforados/síntese química , Proteínas Proto-Oncogênicas pp60(c-src)/química , Domínios de Homologia de src , Animais , Benzamidas/química , Benzamidas/farmacologia , Reabsorção Óssea/tratamento farmacológico , Cristalografia por Raios X , Cicloexanos/química , Cicloexanos/farmacologia , Durapatita/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Modelos Moleculares , Mimetismo Molecular , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Osteoclastos/patologia , Fosfotirosina/química , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , CoelhosRESUMO
Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.
Assuntos
Reabsorção Óssea/metabolismo , Difosfonatos/metabolismo , Osteoclastos/metabolismo , Domínios de Homologia de src/fisiologia , Quinases da Família src/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Dentina/metabolismo , Difosfonatos/química , Difosfonatos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Humanos , Ligantes , Mamíferos , Camundongos , Dados de Sequência Molecular , Osteoclastos/citologia , Osteoporose/metabolismo , Coelhos , Ensaio Radioligante , Ratos , Trítio , Técnicas do Sistema de Duplo-Híbrido , Quinases da Família src/antagonistas & inibidoresRESUMO
Using a novel, solid-phase parallel synthetic route and a computational docking program, a series of phosphorylated nonpeptides were generated to determine their structure-activity relationships (SAR) for binding at the SH2 domain of pp60src (Src). A functionalized benzoic acid intermediate was attached to solid support via Rink amide linkage, which upon acid cleavage generated the desired benzamide template-based nonpeptides in a facile manner. Compounds were synthesized using a combination of solid- and solution-phase techniques. Purification using reversed-phase, semipreparative HPLC allowed for quantitative SAR studies. Specifically, this work focused on functional group modifications, in a parallel fashion, designed to explore hydrophobic binding at the pY+3 pocket of the Src SH2 domain.
Assuntos
Benzamidas/síntese química , Sítios de Ligação , Proteínas Proto-Oncogênicas pp60(c-src)/química , Domínios de Homologia de src , Benzamidas/química , Cristalografia por Raios X , Desenho de Fármacos , Fosforilação , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS: AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS: These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.
Assuntos
Benzoatos/farmacologia , Reabsorção Óssea/prevenção & controle , Inibidores Enzimáticos/farmacologia , Osteoclastos/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Benzoatos/síntese química , Benzoatos/química , Sítios de Ligação/genética , Reabsorção Óssea/etiologia , Reabsorção Óssea/fisiopatologia , Células Cultivadas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Técnicas In Vitro , Ligantes , Camundongos , Dados de Sequência Molecular , Osteoclastos/fisiologia , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Transformação Genética , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de src/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
CD11b/CD18 (Mac-1) is a leukocyte integrin that plays a critical role in neutrophil adhesion and the initiation of acute inflammatory responses. Several Mac-1 blocking mAbs bind to the A-domain of CD11b, a approximately 200 amino acid region in the N-terminal portion of the protein that is involved in ligand binding and Mac-1 functional activity. We examined several CD11b blocking mAbs for different patterns of binding to A-domain. We used human/murine chimeric CD11b expression constructs and deletions of the A-domain to examine binding. We describe the binding characteristics of mAbs 60.1, LM2/1, LPM19C, M170, 44, and 904. All of these mAbs, except for 60.1, bind to the C-terminal half of the human A-domain (CD11b181-316). mAb 60.1 was unique in that it required regions of the N- and C-terminal ends of the A-domain for binding. mAbs 60.1, LPM19C, 904, and 44 all required the A-domain to be intact for binding. This suggests that these CD11b mAbs recognize a conformational epitope. LM2/1 was capable of binding to a fragment of the A-domain, CD11b285-300. Inasmuch as this system has been used to define different mAb binding sites, it may be used to analyze specific ligand binding sites in the A-domain of CD11b.
Assuntos
Antígeno de Macrófago 1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Ligação Competitiva , Linhagem Celular Transformada , Deleção de Genes , Técnicas de Transferência de Genes , Humanos , Antígeno de Macrófago 1/química , Antígeno de Macrófago 1/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de SequênciaRESUMO
Mammalian homeobox genes are widely expressed in the developing central nervous system and are postulated to control developmental processes by regulating gene expression at the transcriptional level. In vitro studies have identified consensus DNA sequences that contain an ATTA core as sites for interaction with homeodomain proteins. Such elements have been found in the upstream regulatory region of the gene encoding beta-amyloid precursor protein, which is associated with the neurological disorder Alzheimer disease. As the beta-amyloid precursor protein gene is also expressed in the developing central nervous system and appears to play a role in cellular regulatory processes, we have examined the possibility that a homeobox gene product can regulate its transcription. We demonstrate by Northern blot analyses and transfection experiments that the expression of the beta-amyloid precursor protein gene is decreased in cultured cells expressing the mouse homeobox gene Hox-3.1.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Genes Homeobox , Animais , Sequência de Bases , Células Cultivadas , Regulação da Expressão Gênica , Células HeLa , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , TransfecçãoRESUMO
We have mapped the mouse peripherin gene, Prph, to chromosome 15 by means of Southern analysis of a panel of Chinese hamster/mouse somatic cell hybrids using a rat peripherin cDNA probe. Peripherin is a recently characterized type III intermediate filament expressed in the peripheral and the central nervous system. Although its exact function is not known, peripherin is likely to be involved in the neuronal cytoskeleton, a role it shares with other intermediate filaments, such as the neurofilament proteins. The intermediate filament gene family is believed to have evolved via gene duplication and dispersal throughout the genome; these processes have resulted in clusters of intermediate filament genes on specific chromosomes and conservation of these chromosomal locations among mammalian species.
Assuntos
Mapeamento Cromossômico , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Animais , Southern Blotting , Sondas de DNA , Células Híbridas , Camundongos , Família Multigênica , PeriferinasRESUMO
Following the discovery of the homeobox as a conserved sequence in developmentally important genes of Drosophila, a plethora of such sequences have been identified in evolutionarily distant organisms. Among mammals, the mouse homeobox genes have been studied most intensively with a hope of deciphering basic mechanisms of embryonic development. The genomic arrangement of many mouse homeobox genes is similar to the organization of the Drosophila genes, suggesting that they arose as a consequence of gene duplication and divergence from a primordial cluster during evolution. Homeobox genes encode proteins that may form a part of the autoregulatory and transregulatory network specifying positional value in the embryo. Supporting this view, the more diverged members of this growing family function as transcription factors, some of which regulate the expression of tissue-specific genes. Mouse homeobox genes are expressed during embryonic development in a spatially restricted manner and alterations in their expression pattern can disrupt embryonic development. The implications of these findings will be discussed in the context of the role of homeobox genes in the embryonic development of Drosophila and other organisms.
Assuntos
Genes Homeobox , Camundongos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Drosophila/embriologia , Drosophila/genética , Regulação da Expressão Gênica , Humanos , Camundongos/embriologia , Dados de Sequência MolecularRESUMO
The human squamous cell carcinoma SqCC/Y1 undergoes spontaneous terminal differentiation in the confluent state. The degree of maturation was markedly increased by glucocorticoids and by both human recombinant and placental lipocortin I. Western analyses demonstrated cellular secretion of lipocortin into the medium. Glucocorticoid-induced maturation was antagonized by a lipocortin I-specific monoclonal antibody, by phospholipase A2 (PLA2), and by arachidonic acid. Induction of the differentiation of SqCC/Y1 cells by lipocortin I was prevented by arachidonic acid. The PLA2 inhibitor, dibromoacetophenone, caused an increase in envelope-competent cells indicating that inhibition of PLA2 results in induction of differentiation. Epidermal growth factor prevented the induction of differentiation by both lipocortin I and by glucocorticoids. The nonsteroidal lipoxygenase/cyclo-oxygenase inhibitor, phenidone, also increased SqCC/Y1 differentiation, suggesting that leukotrienes, thromboxanes, and/or prostaglandins may be involved in lipocortin-mediated regulation of SqCC/Y1 maturation. The findings support a role for lipocortin I in mediating the effects of glucocorticoids on epidermal cell differentiation.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/efeitos dos fármacos , Glucocorticoides/farmacologia , Fosfolipases/antagonistas & inibidores , Anexinas , Anticorpos Monoclonais/imunologia , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Proteínas de Ligação ao Cálcio/imunologia , Carcinoma de Células Escamosas/fisiopatologia , Linhagem Celular , Humanos , Fosfolipases A/farmacologia , Fosfolipases A2 , Células Tumorais Cultivadas/patologiaRESUMO
SqCC/Y1, a human malignant squamous cell carcinoma, spontaneously differentiates when grown to confluence in delipidized serum-containing medium, as measured by the capacity to form detergent-insoluble cornified cell envelopes. Thus, 30% of SqCC/Y1 cells spontaneously attained the differentiated state after 6 days in culture. Exposure of SqCC/Y1 cells to 30, 100, or 300 nM hydrocortisone increased the number of mature cells, producing a 25, 100, and 225%, respective, increase in the number of differentiated cells over the spontaneous rate of maturation. Retinoic acid at levels of 3-300 nM was inhibitory, causing a 24-85% decrease in the number of differentiated cells. Simultaneous treatment with hydrocortisone and retinoic acid indicated mutual antagonism of the effects of these agents on the formation of cornified envelopes. Since hydrocortisone possesses antiangiogenic (AG), mineralocorticoid (MC) and glucocorticoid (GC) activities, steroids with different degrees of GC, MC, and AG potency were examined for their capacities to induce terminal differentiation. Only steroids with GC activity, such as dexamethasone, hydrocortisone, and RU-28362, were capable of increasing the degree of SqCC/Y1 differentiation and antagonizing the inhibitory effects of retinoic acid on the maturation process. In addition, the GC antagonist, RU-38486, reversed the stimulation of cellular differentiation produced by the glucocorticoids. The findings indicate that GC activity is required for the steroid-induced terminal differentiation of SqCC/Y1 cells.
Assuntos
Carcinoma de Células Escamosas/patologia , Diferenciação Celular/efeitos dos fármacos , Glucocorticoides/farmacologia , 17-alfa-Hidroxiprogesterona , Androstanóis/farmacologia , Desoxicorticosterona/farmacologia , Relação Dose-Resposta a Droga , Humanos , Hidrocortisona/farmacologia , Hidroxiprogesteronas/farmacologia , Técnicas In Vitro , Mifepristona/farmacologia , Mineralocorticoides/farmacologia , Neovascularização Patológica , Progesterona/farmacologia , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Differentiation of SqCC/Y1, a human malignant squamous carcinoma, can be modulated by the presence of both corticosteroids and retinoids. To evaluate the regulation of the differentiation of epidermal cells by these agents, we have employed delipidized serum from which glucocorticoids and retinoids were removed. Thirty percent of SqCC/Y1 cells spontaneously expressed the terminally differentiated phenotype after 6 d in culture, as measured by the capacity to form detergent-insoluble cornified envelopes. Exposure of SqCC/Y1 cells to hydrocortisone at concentrations of 30, 100 and 300 nM produced a 25, 100, and 175%, increase, respectively, in the number of differentiated cells over untreated control cultures. Exposure of cells to retinoic acid at levels of from 3 to 300 nM caused a 24 to 85% decrease in the quantity of differentiated cells. Simultaneous treatment of SqCC/Y1 cells with hydrocortisone and retinoic acid resulted in mutual antagonism of cornified envelope formation. Treatment of SqCC/Y1 cells with a 1000-fold molar excess of retinoic acid did not directly alter the uptake of hydrocortisone, and a 100-fold molar excess did not directly inhibit the binding of the corticosteroid to its receptor. Pretreatment of cells for 48, 72, or 96 h with 100 nM retinoic acid decreased the binding of hydrocortisone to its receptor by only 20%, and only resulted in a small decrease in the total amount of hydrocortisone associated with cells 48 h after the addition of retinoic acid. These findings suggest that the antagonism between hydrocortisone and retinoic acid on the terminal differentiation of SqCC/Y1 is not expressed at the level of the corticosteroid receptor.