RESUMO
Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.
Assuntos
Adenoviridae/genética , Vetores Genéticos , Transgenes , Vacinas Virais/genética , Adenoviridae/fisiologia , Sequência de Bases , Linhagem Celular , Primers do DNA , DNA Viral/genética , DNA Viral/isolamento & purificação , Humanos , Replicação ViralRESUMO
OBJECTIVE: This study was undertaken to determine the effect of nitric oxide (NO) on tissue factor (TF) expression in vascular smooth muscle cells. STUDY DESIGN: Rat aortic smooth muscle cells (RASMCs) were exposed to NO delivered exogenously with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or produced endogenously after infection with an adenoviral vector carrying human inducible NO synthase (AdiNOS). Functional TF activity was assessed with chromogenic TF assay. TF antigen was determined with immunohistochemistry. Northern blot analysis was used to determine steady- state TF messenger RNA (mRNA). Electrophoretic mobility gel shift assay was performed to determine the nuclear binding activity of nuclear factor kappa-B (NFkappaB). NFkappaB activity was inhibited by either prior transduction of RASMCs with mutant IkappaB or treatment with pyrrolidine dithiocarbamate. RESULTS: RASMCs exposed to SNAP or infected with AdiNOS exhibited increased functional TF activity and antigen. Regardless of the source of NO, a time-dependent and concentration-dependent increase in TF activity was observed. Steady-state TF mRNA levels were also increased by NO delivered via either method. NFkappaB nuclear binding activity was also increased by NO. Inhibition of NFkappaB activity by either pyrrolidine dithiocarbamate treatment or mutant IkappaB transduction abrogated NO-induced enhancement of TF mRNA and functional activity. CONCLUSION: In RASMC, NO exposure results in upregulation of TF functional activity, antigen, and mRNA. This effect appears to be mediated by an NFkappaB-dependent pathway.
Assuntos
Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiologia , Tromboplastina/metabolismo , Adenoviridae , Animais , Antígenos/metabolismo , Aorta Torácica , Bioensaio , Northern Blotting , Células Cultivadas , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Vetores Genéticos , Proteínas I-kappa B/genética , Proteínas I-kappa B/farmacologia , Imuno-Histoquímica , Masculino , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ligação Proteica , Pirrolidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina/farmacologia , Tiocarbamatos/farmacologia , Tromboplastina/genética , Tromboplastina/imunologia , Transfecção , Regulação para CimaRESUMO
The functional role of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell (VSMC) apoptosis remains unknown. In this study, VSMC from p53-/- and p53+/+ murine aortas were exposed to exogenous or endogenous sources of NO. Unexpectedly, p53-/- VSMC were much more sensitive to the proapoptotic effects of NO than were p53+/+ VSMC. Furthermore, this paradox appeared to be specific to NO, because other proapoptotic agents did not demonstrate this differential effect on p53-/- cells. NO-induced apoptosis in p53-/- VSMC occurred independently of cGMP generation. However, mitogen-activated protein kinase (MAPK) pathways appeared to play a significant role. Treatment of the p53-/- VSMC with S-nitroso-N-acetylpenicillamine resulted in a marked activation of p38 MAPK and, to a lesser extent, of c-Jun NH(2)-terminal kinase, mitogen-activated protein kinase kinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase, ERK). Furthermore, basal activity of the MEK-p42/44 (ERK) pathway was increased in the p53+/+ VSMC. Inhibition of p38 MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-induced apoptosis. Therefore, p53 may protect VSMC against NO-mediated apoptosis, in part, through differential regulation of MAPK pathways.
