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Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1α and its constitutive β subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophorelabeled HIF-1 subtmits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1α than of HIF-1β, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between α and β subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models. [ABSTRACT FROM AUTHOR]

The effect of natural and recombinant interferons (nIFN, rIFN) on cell growth, apoptosis and the production of vascular endothelial growth factor (VEGF) was investigated in the human melanoma cell line IGR 1. We determined cell proliferation, cell vitality, DNA synthesis, apoptosis, intracellular oxygen radicals (ROS) and VEGF-mRNA as well as VEGF-protein levels. rIFN-γ significantly inhibited growth by decreasing DNA synthesis and increasing apoptosis. Less pronounced was the growth inhibitory effect of nIFN-β because an increased rate of apoptosis was outweighed by enhanced DNA synthesis. nIFN-α only had minor effects on cell growth parameters. Under long-term incubation (144 h) nIFN-β decreased, but rIFN-γ increased production of the angiogen VEGF. Our data underscore the multiple effects of IFNs on melanoma cells and may contribute to the understanding of ambivalent results of melanoma therapy by IFNs. Particularly, the increased VEGF production under long-term treatment with serum IFN levels between 100 and 1200 IU/ml should be kept in mind. [ABSTRACT FROM AUTHOR]

Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 μM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 μM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three-dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The OH levels decreased in cobalt-treated (to 81 %) as well as in nickel-treated (to 67 %) HepG2 cells, accompanied by an increase of EPO expression to 167 % and 150 %, respectively. Our results underline the importance of OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins. [ABSTRACT FROM AUTHOR]