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Purpose: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. Materials and methods: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm[sup -2]) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. Results: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. Conclusion: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways. [ABSTRACT FROM AUTHOR]

The recently reported influence of proinsulin C-peptide on insulin prompted us to examine structural features of the C-peptide. Four sets of limited pattern similarities towards inter-chain end regions of insulin were noticed, involving secondary structure elements, binding residues and intra- as well as inter-peptide residue similarities. Using surface plasmon resonance, we examined insulin binding to truncated, soluble insulin receptor A and IGF-1 receptor, but C-peptide effects on these bindings were not detectable. Two forms of the insulin receptor, differing in activation of gene transcription with regards to (pre)proinsulin and glucokinase, respectively, were also uninfluenced by C-peptide. We conclude that the pattern similarities, if functional, reflect C-peptide interactions with molecules other than both insulin A and B receptors and IGF-1 receptors. Any such effects are of interest in relation to reported binding interactions between insulin and C-peptide. [ABSTRACT FROM AUTHOR]

Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon stimulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell. [ABSTRACT FROM AUTHOR]