PubMed · 8202128
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D J McCance. 1994-06-09. Gene safety.. https://doi.org/10.1038/369436d0
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A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.
Dendritic cells (DC) are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen (TAA) or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection (MOI). In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein (EGFP)-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 x g at 37 degrees C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity (MFI) of EGFP expression was about 150 (at 37 degrees C for 2 h). With centrifugal transduction (2000 x g at an MOI of 50 at 37 degrees C for 2 h), 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced (MFI: about 3100 or greater). Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction (MLR). The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC.
Nitric oxide (NO) can modulate numerous genes through several pathways, yet some genes may be modulated only in the presence of the inflammatory stimuli that upregulate the inducible nitric oxide synthase (iNOS) rather than by NO alone. Furthermore, the role of prior expression of iNOS in the modulation of genes by NO is unknown. We addressed these issues in hepatocytes harvested from iNOS-null (iNOS(-/-)) mice exposed to NO by treatment with NO donors or by infection with an adenovirus-expressing human iNOS (Ad-iNOS), rather than by stimulation with inflammatory cytokines. Differential display and gene array analyses performed on mRNA derived from iNOS(-/-) hepatocytes demonstrated that infection with Ad-iNOS, but not infection with a control adenovirus expressing the beta-galactosidase gene (Ad-LacZ), induced a gene fragment identical to cytochrome P450 2E1 (CYP2E1). Northern analysis performed with this fragment demonstrated that treatment of iNOS(-/-) hepatocytes with Ad-iNOS or with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not control treatment or infection with Ad-LacZ, resulted in increased expression of CYP2E1. Inhibition of soluble guanylyl cyclase partially blocked the induction of CYP2E1 mRNA by Ad-iNOS. Rat hepatocytes treated with SNAP also exhibited increased expression of CYP2E1 mRNA. Preliminary studies, however, suggest that the induction of CYP2E1 in the rat hepatocytes treated with cytokines was not reduced in the presence of a NOS inhibitor. Our results suggest that CYP2E1 can be induced solely by NO derived from iNOS, at least partly in a cyclic GMP-dependent manner and independently of inflammatory stimuli or of prior exposure to NO.