RESUMO
OBJECTIVE: Preclinical data show that exogenous administration of recombinant human bone morphogenetic protein-2 (rhBMP-2) to human oral carcinoma cell lines increases pathogenicity using a nude mouse model. The objectives of this study are to (1) describe the characteristics of baseline protein expression of BMP-2 in head and neck squamous cell carcinomas (HNSCC) and (2) determine if BMP-2 expression level correlates with worse oncologic outcomes. STUDY DESIGN: Retrospective analysis of previously harvested patient samples. SETTING: Academic medical center. SUBJECTS: In total, 149 patients with oral cavity, oropharynx, larynx, and hypopharynx HNSCC treated between January 1, 1997, and December 31, 2004. METHODS: A tissue microarray of HNSCC was assembled and immunohistochemistry for BMP-2 performed. Staining was quantified using a standardized scoring system. Specimens were dichotomized into high or low expression level. Statistical analyses using log-rank, Wilcoxon, and Fisher exact test were performed for associations between BMP-2 protein level and clinicopathologic features and patient survival. RESULTS: BMP-2 expression at any level was noted in 146 of 149 (98%) of samples. Tumors with high BMP-2 expression had higher rates of local failure compared with low-expressing tumors (17.3% vs 6.3%; P = .04). There was no significant association for BMP-2 expression level with tumor location, T stage, N stage, overall survival, regional failure, or distant failure. CONCLUSION: Head and neck squamous cell carcinomas with high baseline BMP-2 protein level are associated with higher rates of local recurrence. These data have important implications for using rhBMP-2 in tissue engineering reconstructive approaches in the setting of cancer-related defects.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Hipofaríngeas , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Laríngeas , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/metabolismo , Neoplasias Orofaríngeas/metabolismo , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Análise Serial de TecidosRESUMO
Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and γ-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Diferenciação Celular/genética , Células Eritroides/citologia , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Antígenos CD34/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Citarabina/farmacologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562 , Lentivirus/genética , Leucemia/genética , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Interferência de RNA , Transdução GenéticaRESUMO
OBJECTIVES/HYPOTHESIS: To establish the relevance of the bone morphogenetic protein (BMP) signaling pathway in human oral squamous cell carcinoma (OSCCA) cell lines and determine if there is a biologic impact of stimulating this pathway with recombinant human (rh) BMP-2. STUDY DESIGN: In vitro laboratory investigations and in vivo analysis using an orthotopic animal model for oral cancer. METHODS: Gene expression profiles for BMP-2 and components of the BMP-signaling pathway were determined using reverse transcriptase-polymerase chain reaction. In vivo effects were evaluated using Kaplan-Meier survival analysis and studying histopathologic changes in established tumor xenografts with or without rhBMP-2 pretreatment. A phosphokinase array was used to detect levels of activation in signaling kinases. RESULTS: The BMP-2 gene was expressed in 90% of the 30 OSCCA cell lines tested. Gene expression of all components of the BMP-signaling pathway was highly conserved. Tumor xenografts established with rhBMP-2-treated cells showed more rapid local growth that resulted in worse animal survival as compared to the control group. These tumors had a more poorly differentiated morphology. Changes in protein kinases suggested interactions of BMP-2 signaling with the Wnt-ß-catenin, and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways. CONCLUSIONS: Human OSCCA cell lines frequently express BMP-2 and all necessary components of the BMP-signaling pathway. Exogenous treatment of human OSCCA cell lines with rhBMP-2 prior to engraftment in an orthotopic animal model caused the subsequent tumors to be more locally aggressive with worse survival. Continued caution should be used for considering rhBMP-2 for reconstruction of bone defects in oral cancer patients.
Assuntos
Proteína Morfogenética Óssea 2/efeitos adversos , Carcinoma de Células Escamosas/induzido quimicamente , Neoplasias Bucais/induzido quimicamente , Fator de Crescimento Transformador beta/efeitos adversos , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Humanos , Camundongos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Transplante de Neoplasias , Proteínas Recombinantes/efeitos adversos , Células Tumorais CultivadasRESUMO
OBJECTIVE: To determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) has biological effects on the invasiveness of human oral squamous cell carcinoma (OSCCA) cell lines. STUDY DESIGN: Laboratory investigation using six human OSCCA cell lines, with three cell lines having baseline gene expression of BMP-2 and three cell lines without baseline gene expression of BMP-2. METHODS: The invasiveness of each cell line was measured using a matrigel invasion assay with or without stimulation by rhBMP-2. A tumor metastasis quantitative PCR array was used to establish whether observed findings from the invasion assay correlated to changes in gene expression. RESULTS: There was a significant increase in tumor cell invasion in response to rhBMP-2 in all BMP-2 positive cell lines but no change in the cell lines that did not express the BMP-2 gene. Quantitative PCR revealed that changes in gene expression were distinctly different based on the baseline gene expression of BMP-2 and favored a more metastatic genotype in the BMP-2-positive cells. CONCLUSIONS: Recombinant human BMP-2 has an adverse biological effect on invasiveness of human OSCCA cell lines in vitro. This adverse effect is dependent on the baseline gene expression of BMP-2. Changes in expression of genes involved with tumor metastasis correlated to the invasion assay findings. These data raise concern for the safe application of rhBMP-2 for reconstruction of bone defects in oral cancer patients.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Fator de Crescimento Transformador beta/farmacologia , Proteína Morfogenética Óssea 2/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Bucais/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/genéticaRESUMO
Acute insulin resistance can develop following critical illness and severe injury, and the mortality of critically ill patients can be reduced by intensive insulin therapy. Thus, compensating for the insulin resistance in the clinical care setting is important. However, the molecular mechanisms that lead to the development of acute injury/infection-associated insulin resistance are unknown, and the development of acute insulin resistance is much less studied than chronic disease-associated insulin resistance. An animal model of injury and blood loss was utilized to determine whether acute skeletal muscle insulin resistance develops following injury, and surgical trauma in the absence of hemorrhage had little effect on insulin-mediated signaling. However, following hemorrhage, there was an almost complete loss of insulin-induced Akt phosphorylation in triceps, and severely decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1. The severity of insulin resistance was similar in triceps and extensor digitorum longus muscles, but was more modest in diaphragm, and there was little change in insulin signaling in cardiac muscle following hemorrhage. Since skeletal muscle is an important insulin target tissue and accounts for much of insulin-induced glucose disposal, it is important to determine its role in injury/infection-induced hyperglycemia. This is the first report of an acute development of skeletal muscle insulin signaling defects. The presented data indicates that the defects in insulin signaling occurred rapidly, were reversible and more severe in some skeletal muscles, and did not occur in cardiac muscle.
Assuntos
Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Diafragma/metabolismo , Hemorragia/metabolismo , Hemorragia/fisiopatologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Miocárdio/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein (approximately 50%) present in mature myelin from the central nervous system (CNS). Plp gene activity is low to nonexistent early in development but sharply increases, concurrently with the active myelination period of CNS development. Work from our laboratory suggests that the temporal regulation of Plp gene expression in mice is mediated by a positive regulatory element located within Plp intron 1 DNA. We have termed this regulatory element/region ASE (for antisilencer/enhancer). The ASE is situated approximately 1 kb downstream of exon 1 DNA and encompasses nearly 100 bp. To understand the mechanisms by which the ASE augments Plp gene expression in oligodendrocytes, Plp-lacZ constructs were generated and transfected into a mouse oligodendroglial cell line (N20.1). Results presented here demonstrate that upstream regulatory elements in the Plp promoter/5'-flanking DNA are not required for ASE activity; the ASE worked perfectly well when the thymidine kinase (TK) promoter was substituted for the Plp promoter. However, the relative location of the ASE appears to be important. When placed upstream of 2.4 kb of Plp 5'-flanking DNA, or downstream of the lacZ expression cassette, the ASE was no longer effective. Thus, the ASE might have to be in the context of the intron in order to function. To begin to identify the crucial nucleotides within the ASE, orthologous sequences from rat, human, cow, and pig Plp genes were swapped for the mouse sequence. Results presented here demonstrate that the orthologous sequence from rat can substitute for the mouse ASE, unlike those from human, cow, or pig.
Assuntos
DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Oligodendroglia/metabolismo , Região 5'-Flanqueadora/genética , Animais , Bovinos , Linhagem Celular Transformada , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sequência Conservada/genética , DNA/isolamento & purificação , Genes Reporter/genética , Humanos , Íntrons/genética , Óperon Lac/genética , Camundongos , Bainha de Mielina/genética , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Regiões Promotoras Genéticas/genética , Ratos , Homologia de Sequência do Ácido Nucleico , Sus scrofa , TransfecçãoRESUMO
The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein found in mature CNS myelin. Expression of the gene is dynamic and peaks during the active myelination period of CNS development. The surge in Plp gene activity during this period has been purported to be mediated by a positive regulatory region located within the first intron. This region, designated ASE for antisilencer/enhancer, is located approximately 1 kb downstream of exon 1 sequences and encompasses nearly 100 bp. However, neither the critical nucleotides within this region, nor the associated DNA-binding proteins have been identified. In the present study, DNase I footprinting analysis demonstrated widespread protection of the region on both the coding and non-coding strands suggesting that multiple transcription factors are likely involved. Targeting of putative DNA-protein binding sites contained within the ASE by gel shift, transfection and mutagenesis studies revealed the importance of several AP-1-like binding sites in governing high levels of Plp gene expression in oligodendrocytes. Our results suggest that factors, which bind to these sites, form the core of a multiprotein complex that assembles on the ASE and ultimately affects the temporal regulation of the gene in oligodendrocytes.
Assuntos
Regulação da Expressão Gênica , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Oligodendroglia/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Pegada de DNA/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Elementos Facilitadores Genéticos , Fígado , Camundongos , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transfecção/métodosRESUMO
The myelin proteolipid protein gene (Plp ) is expressed primarily in oligodendrocytes. Yet how the gene remains repressed in nonexpressing cells has not been defined, and potentially could cause adverse effects in an organism if the mechanism for repression was impaired. Previous studies suggest that the first intron contains element(s), which suppress expression in nonexpressing cells, although the identity of these elements within the 8 kb intron was not characterized. Here we report the localization of multiple negative regulatory elements that repress Plp gene expression in nonexpressing cells (+/+ Li). Two of these elements (regions) correspond to those used by Plp expressing cells (N20.1), whilst another acts in a cell type-specific manner (i.e. operational in +/+ Li liver cells, but not N20.1 cells). By gel-shift and DNase I footprinting analyses, the factor(s) that bind to the cell type-specific negative regulatory region appear to be far more abundant in +/+ Li cells than in N20.1 cells. Thus, Plp gene repression is mediated through the combinatorial action of both "general" and cell type-specific negative regulatory elements. Additionally, repression in +/+ Li cells cannot be overcome via an antisilencer/enhancer element, which previously has been shown to function in N20.1 cells.