UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation-and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and͞or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.
Most techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5-bromodeoxyuridine (BrdUrd) offers such a potential. We illustrate here that the bivariate analysis of nonBrdUrd-quenched ethidium bromide vs. BrdUrd-quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.
Key terms: bromodeoxyuridine, Hoechst dyeCell cycle, flow cytometry, Accurate determination of cell proliferation is pertinent to clinical and experimental immunology and oncology, as well as to many facets of experimental cell biology. Classical univariate DNA flow cytometry is limited in its analytical power as it cannot distinguish between cells that have undergone multiple divisions within a given observation period. This constraint holds also for the more recent bivariate fluorescence techniques using either acridine orange staining (1) or bromodeoxyuridine(BrdUrd)-antibody staining (2). In contrast, continuous labeling with BrdUrd during multiple rounds of DNA synthesis has been shown to distinguish chromatids and cell fractions that belong to successive cell cycles (3,7,8,13). Such continuous labeling with BrdUrd combined with univariate Hoechst 33258 flow cytometry has initially been used to analyze the replicative heterogeneity within primary cell cultures of human origin (4,5,7,14). Here we show that extension of this principle to bivariate analysis of (nonquenched) ethidium bromide vs. (quenched) Hoechst 33258 fluorescence improves the analytical power of the continuous BrdUrd-labeling method. In particular, we wish to document that application of this improved technique is by no means restricted to the analysis of primary cell cultures of human origin.
MATERIALS AND METHODSHuman diploid fibroblast-like cells (HDF) were cultured in minimum essential medium (MEM) supplied with 10% fetal bovine serum (FBS); NIH-3T3 cells were grown in a mixture of equal amounts of MEM and Ham's F-10 (Gibco), including 10% FBS. Ficoll-separated human peripheral blood lymphocytes (HPBL), human Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (HLBC), and murine splenic B-lymphocytes (MSL) were cultured in RPMI-1640 medium supplemented with 10% or 15% FBS. HPBL and MSL cells are naturally quiescent populations. HLBC cells enter proliferative quiescence if they are left unfed for more than 4 days. HDF cells were rendered quiescent by serum depletion of confluent cultures (48 h; 0.1% FBS). NIH-3T3 cells do not tolerate such low serum concentrations; they were rendered quiescent by 24-h exposures to 0.5% FBS. Quiescent HDF cells were stimulated by trypsinization and dilution in MEM containing 10% FBS and equimolar amounts of BrdUrd and deoxycytidine (dC) (65 pM), whereas NIH-3T3 cells were trypsinized and...
In adult rats, acute nicotine, the major psychoactive ingredient in tobacco smoke, stimulates the hypothalamic-pituitary-adrenal axis (HPA), resulting in activation of brain areas involved in stress and anxiety-linked behavior. However, in rat pups the first two postnatal weeks are characterized by hypo-responsiveness to stress, also called the 'stress non-responsive period' (SNRP). Therefore, we wanted to address the question if acute nicotine stimulates areas involved in the stress response during SNRP? To determine neuronal activation, the expression of the immediate-early genes c-fos and Arc was studied in the central nucleus of the amygdala (CeA), bed nucleus stria terminalis (BST) and paraventricular hypothalamic nucleus (PVN), which are areas involved in the neuroendocrine and central stress response. Rat pups received nicotine tartrate (2 mg/kg) or saline by i.p. injection at postnatal day (P) 5, 7 and 10 and their brains were removed after 30 min. We used semi-quantitative radioactive in situ hybridization with gene specific antisense cRNA probes in coronal sections. In control pups, c-fos expression was low in most brain regions, but robust Arc hybridization was found in several areas including cingulate cortex, hippocampus and caudate. Acute nicotine resulted in significant induction of c-fos expression in the PVN and CeA at P5, P7 and P10, and in the BST at P7 and P10. Acute nicotine significantly induced expression of Arc in CeA at P5, P7 and P10, and in the BST at P10. In conclusion, acute nicotine age dependently activated different brain areas of the HPA axis during the SNRP. After P7, the response was more pronounced and included the BST, suggesting differential maturation of the HPA axis in response to nicotine.
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