Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance transporter P-glycoprotein (P-gp) has not been investigated. Herein, we demonstrate that with increasing size of DU-145 prostate multicellular tumor spheroids the pericellular oxygen pressure and the generation of reactive oxygen species decreased, whereas the alpha-subunit of HIF-1 (HIF-1alpha) and P-gp were up-regulated. Furthermore, P-gp was up-regulated under experimental physiological hypoxia and chemical hypoxia induced by either cobalt chloride or desferrioxamine. The pro-oxidants H2O2 and buthionine sulfoximine down-regulated HIF-1alpha and P-gp, whereas up-regulation was achieved with the radical scavengers dehydroascorbate, N-acetylcysteine, and vitamin E. The correlation of HIF-1alpha and P-gp expression was validated by the use of hepatoma tumor spheroids that were either wild type (Hepa1) or mutant (Hepa1C4) for aryl hydrocarbon receptor nuclear translocator (ARNT), i.e., HIF-1beta. Chemical hypoxia robustly increased HIF-1alpha as well as P-gp expression in Hepa1 tumor spheroids, whereas no changes were observed in Hepa1C4 spheroids. Hence, our data demonstrate that expression of P-gp in multicellular tumor spheroids is under the control of HIF-1.

In higher organisms, respiratory and cardiovascular systems provide and appropriately distribute oxygen (O 2) to tissues and cells to serve as the terminal electron acceptor during mitochondrial oxidative phosphorylation, which is the major biochemical reaction for generating energy in the form of ATP. The process of extracting oxygen from the environment and its distribution, not only for oxidative phosphorylation but also as a substrate for other biochemical reactions, has been conserved through evolution by the development of advanced multi-level systems, which tightly maintain O2 homeostasis, i.e. keep the O 2 concentration, even within a single cell, within a narrow physiological range, allowing the cell to survive, function and thrive in regions with a heterogeneous PO∑ distribution. Under physiological conditions the arterial PO∑ is about 90·mmHg. However, differences in vascularization, tissue diffusion properties and cell-specific oxygen consumption most likely account for the heterogeneous PO∑ distribution seen within the brain, resulting in tissue PO∑ levels from 90·mmHg down to These various responses might be based on a range of oxygen-sensing signal cascades, including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low P O∑ affinity cytochrome c oxidase (aa3) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF prolyl-hydroxylase (PHD) and HIF asparaginyl hydroxylase, known as factor-inhibiting HIF (FIH-1). Thus specific oxygen-sensing cascades, by means of their different oxygen sensitivities, cell-specific and subcellular localization, may help to tailor various adaptive responses according to differences in tissue oxygen availability.

SummaryTwo-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-alpha activity and plasma membrane potassium-channel (PMPC) conductivity.

SummaryTwo-photon (TP) excitation (820 -1150 nm) and emission (280 -700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their onephoton (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.