RESUMO
The cyclic AMP-dependent protein kinase inhibitor (PKI) mRNA and protein are negatively and tissue-specifically regulated in the kidney by 1 alpha, 25(OH)2D3. A 17-kb PKI clone, isolated from a chick genomic library, revealed that the PKI gene consists of two exons separated by a 4.5-kb intron. A 411-bp upstream region (constituting 93 bp upstream and 318 bp downstream from the transcriptional start site) containing a putative negative VDRE (nVDRE) fused to the luciferase gene was used for transient transfections of primary cultures of chick kidney cells. Luciferase activity was significantly down-regulated in response to 1 alpha, 25(OH)2D3. This result suggests that the promoter region containing the putative nVDRE plays a pivotal role in the negative regulation of PKI gene transcription.
Assuntos
Calcitriol/farmacologia , Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intracelular , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Galinhas , Clonagem Molecular , DNA Complementar/química , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/metabolismo , Luciferases/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transfecção , beta-Galactosidase/genéticaRESUMO
UNLABELLED: Human pancreatic ductal adenocarcinomas overexpress transforming growth factor-betas (TGF-betas). This overexpression has been correlated with decreased patient survival. TGF-betas bind to a type II TGF-beta receptor (TbetaRII) dimer, which heterotetramerizes with a type I TGF-beta receptor (TbetaRI) dimer, thereby activating downstream signaling. PURPOSE AND EXPERIMENTAL DESIGN: To determine whether blocking TGF-beta actions would suppress pancreatic cancer cell growth in vivo, we expressed a soluble TbetaRII, encoding amino acids 1-159 of the extracellular domain in COLO-357 human pancreatic cancer cells. This cell line expresses all of the three mammalian TGF-beta isoforms and is growth inhibited by TGF-beta in vitro. RESULTS: COLO-357 clones expressing soluble TbetaRII were no longer growth inhibited by exogenous TGF-beta1 and exhibited a marked decrease in their invasive capacity in vitro. When injected s.c. into athymic mice, these clones exhibited attenuated growth rates and angiogenesis and decreased levels of plasminogen activator inhibitor-1 mRNA as compared with tumors formed by sham-transfected cells. CONCLUSIONS: These results indicate that endogenous TGF-betas can confer a growth advantage in vivo to a pancreatic cancer cell line that is growth inhibited in vitro and suggest that a soluble receptor approach can be used to block these tumorigenic effects of TGF-betas.
Assuntos
Neoplasias Pancreáticas/terapia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Movimento Celular/efeitos dos fármacos , Feminino , Expressão Gênica , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Solubilidade , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Six A-ring analogs of 1alpha,25-dihydroxyvitamin D3 (1, 1alpha,25-(OH)2-D3) 3-deoxy-3-thia-1alpha,25-(OH)2-D3 (3), 3-deoxy-3-thia-1alpha,25-(OH)2-D3-3alpha-oxide (6), 3-deoxy-3-thia-1alpha,25-(OH)2-D3-3beta-oxide (7) and the 5,6-trans counterparts 5, 8, and 9, respectively--were tested for their ability to inhibit 25-hydroxy-D3-1alpha-hydroxylase (1-OH-ase) in vitro in mitochondria isolated from kidneys of vitamin D deficient chicks. The six analogs were also evaluated in terms of their ability to bind to the chicken intestinal nuclear receptor (VDR) in comparison to the natural hormone 1alpha,25-(OH)2-D3. Analog 7 is not only the best inhibitor of the 1-OH-ase but it also binds effectively to the chick intestinal receptor. It is established that vitamin D analogs must have a 1alpha oxygen group for effective inhibition of the 1-OH-ase. This functional group is also needed for effective binding to the chick intestinal VDR.