Elmar Krause

Overcoming Intrinsic Multidrug Resistance in Melanoma by Blocking the Mitochondrial Respiratory Chain of Slow-Cycling JARID1Bhigh Cells

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

Depletion of intracellular calcium stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.

Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes “capacitative calcium entry” in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance.

Mitochondria Positioning Controls Local Calcium Influx in T Cells

Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function, it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at stable ISs in Th cells.

Comparison of 3 spectrophotometric methods for carotenoid determination in frequently consumed fruits and vegetables.

UNLABELLED Carotenoids are C-40 tetraterpenoid compounds with potential health beneficial effects. Major dietary sources include a variety of fruits and vegetables. Rapid screening methods are therefore desired, but their accuracy varies depending on the carotenoid profile and the matrix of the plant food. In the present study, 3 different methods were compared, all based on a rapid extraction protocol and spectrophotometric measurements to determine the total amount carotenoids present in fruits and vegetables (n = 28), either with or without chlorophyll. Published methods (a) Lichtenthaler and (b) Hornero-Méndez and Mínguez-Mosquera were compared with a newly developed method (method c) based on the average molar absorption coefficient (135310 Lcm(-1)mol(-1)) and wavelength (450 nm in acetone), for the 5 predominant carotenoid species (beta-carotene, zeaxanthin, lycopene, lutein, beta-cryptoxanthin) in the investigated foods. All results were compared to HPLC (method d). To avoid overestimating carotenoid concentrations due to chlorophyll A and B presence, the effect of saponification was studied for all methods. Overall, saponification led to significant carotenoid losses (12.6 +/- 0.9%). Methods a, b, c, and d yielded 5.1 +/- 0.4 mg/100 g, 4.6 +/- 0.5 mg/100 g, 4.3 +/- 0.5 mg/100 g, and 4.2 +/- 0.5 mg/100 g total carotenoids, respectively, with method a leading to significant higher mean concentrations compared to all other methods (P < 0.001, Bonferroni) with methods b and c being not significantly different and highly correlated compared to HPLC (> r = 0.95). Similar results were found when stratifying for chlorophyll content and fruits compared with vegetables, however, accuracy varied for individual fruits, highlighting the limitation to use the same method for all plant foods. PRACTICAL APPLICATION This study presents a comparison of various rapid spectrophotometric measurements to determine total carotenoid content in various fruits and vegetables and could aid in the selection of the appropriate method for individual plant foods with different carotenoid profile and matrices.

Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Hormonal Control of ADP-ribosyl Cyclase Activity in Pancreatic Acinar Cells from Rats

Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the β-NAD+ analogs 1,N6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP.

Divalent Minerals Decrease Micellarization and Uptake of Carotenoids and Digestion Products into Caco-2 Cells

Carotenoids are lipophilic, dietary antioxidants with the potential to prevent chronic and age-related diseases. Prior to their availability for physiological functions, carotenoids require micellarization and intestinal uptake, both constituting marginally understood processes. Based on an in vitro digestion model coupled to Caco-2 cells, we assessed the effect of dietary abundant divalent ions on spinach-derived carotenoid micellarization and cellular uptake: Ca and Mg ranging from 7.5 to 25 mmol/L in the digesta and Zn and Fe ranging from 3.8 to 12.5 mmol/L. Both micellarization and uptake were significantly inhibited by minerals in a concentration-dependent manner, with stronger effects for Fe and Zn compared to Ca and Mg. Compared to controls (no mineral addition), fractional micellarization and uptake were decreased to the greatest extent (to 22.5 and 5.0%, respectively; P < 0.001) by 12.5 mmol/L Fe. Effects of Mg were of the least magnitude; at 25 mmol/L, only uptake was decreased significantly to 69.2% of the control value (P < 0.001). Total cellular carotenoid uptake from test meals decreased similarly compared to micellarization; however, decreased β-carotene micellarization was counterbalanced by improved fractional cellular uptakes from the micelles for all ions. Compared to controls, fractional β-carotene uptake from the micelles was greater in samples digested in the presence of Fe, Ca, and Zn, by up to 5-10 times at the highest concentrations of each ion (P < 0.001). Like for the above carotenoids, a high cellular uptake of the epoxycarotenoid conversion products neochrome (from neoxanthin) and luteoxanthin+auroxanthin (from violaxanthin) was also observed. The present results indicate that divalent ions may inhibit carotenoid micellarization and uptake.

Low Cytoplasmic [Ca2+] Activates ICRACIndependently of Global Ca2+ Store Depletion in RBL-1 Cells

Release of Ca2+ from inositol (1,4,5)-trisphosphate-sensitive Ca2+ stores causes “capacitative calcium entry,” which is mediated by the so-called “Ca2+ release-activated Ca2+ current” (ICRAC) in RBL-1 cells. Refilling of the Ca2+stores or high cytoplasmic [Ca2+] ([Ca2+]cyt) inactivate ICRAC. Here we address the question if also [Ca2+]cyt lower than the resting [Ca2+]cyt influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both ICRAC and [Ca2+] within Ca2+ stores of RBL-1 cells ([Ca2+]store). We found that low [Ca2+]cyt in the range of 30–50 nm activates ICRAC and Ca2+ influx spontaneously and independently of global Ca2+ store depletion, while elevation of [Ca2+]cyt to the resting [Ca2+]cyt (100 nm) resulted in store dependence of ICRAC activation. We conclude that spontaneous activation of ICRAC by low [Ca2+]cyt could serve as a feedback mechanism keeping the resting [Ca2+]cyt constant.

Cell Side-specific Sensitivities of Intracellular Ca2+ Stores for Inositol 1,4,5-Trisphosphate, Cyclic ADP-ribose, and Nicotinic Acid Adenine Dinucleotide Phosphate in Permeabilized Pancreatic Acinar Cells from Mouse

In pancreatic acinar cells hormonal stimulation leads to a cytosolic Ca2+ wave that starts in the apical cell pole and subsequently propagates toward the basal cell side. We used permeabilized pancreatic acinar cells from mouse and the mag-fura-2 technique, which allows direct monitoring of changes in [Ca2+] of intracellular stores. We show here that Ca2+ can be released from stores in all cellular regions by inositol 1,4,5-trisphosphate. Stores at the apical cell pole showed a higher affinity to inositol 1,4,5-trisphosphate (EC50 = 89 nm) than those at the basolateral side (EC50 = 256 nm). In contrast, cADP-ribose, a modifier of Ca2+-induced Ca2+ release, and nicotinic acid adenine dinucleotide phosphate (NAADP) were able to release Ca2+ exclusively from intracellular stores located at the basolateral cell side. Our data agree with observations that upon stimulation Ca2+ is released initially at the apical cell side and that this is caused by high affinity inositol 1,4,5-trisphosphate receptors. Moreover, our findings allow the conclusion that in Ca2+ wave propagation from the apical to the basolateral cell side observed in pancreatic acinar cells Ca2+-induced Ca2+ release, modulated by cADP-ribose and/or NAADP, might be involved.

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion

Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.