Controlled Frustration Release on the Kagome Lattice by Uniaxial-Strain Tuning.

It is predicted that strongly interacting spins on a frustrated lattice may lead to a quantum disordered ground state or even form a quantum spin liquid with exotic low-energy excitations. However, a controlled tuning of the frustration strength, separating its effects from those of disorder and other factors, is pending. Here, we perform comprehensive ^{1}H NMR measurements on Y_{3}Cu_{9}(OH)_{19}Cl_{8} single crystals revealing an unusual Q[over →]=(1/3×1/3) antiferromagnetic state below T_{N}=2.2 K. By applying in situ uniaxial stress, we break the symmetry of this disorder-free, frustrated kagome system in a controlled manner yielding a linear increase of T_{N} with strain, in line with theoretical predictions for a distorted kagome lattice. In-plane strain of ≈1% triggers a sizable enhancement ΔT_{N}/T_{N}≈10% due to a release of frustration, demonstrating its pivotal role for magnetic order.

Evidence that atypical protein kinase C-lambda and atypical protein kinase C-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

Human diabetes is associated with hyperreactivity of vascular smooth muscle cells due to altered subcellular Ca2+ distribution.

Alterations of vascular smooth muscle function have been implicated in the development of vascular complications and circulatory dysfunction in diabetes. However, little is known about changes in smooth muscle contractility and the intracellular mechanisms contributing to altered responsiveness of blood vessels of diabetic patients. Therefore, smooth muscle and endothelial cell function were assessed in 20 patients with diabetes and compared with 41 age-matched control subjects. In rings from uterine arteries, smooth muscle sensitivity to K+, norepinephrine (NE), and phenylephrine (PE) was enhanced by 1.4-, 2.3-, and 9.7-fold, respectively, and endothelium-dependent relaxation was reduced by 64% in diabetic patients, as compared with control subjects. In addition, in freshly isolated smooth muscle cells from diabetic patients, an increased perinuclear Ca2+ signaling to K+ (30 mmol/l >73%; 60 mmol/l >68%) and NE (300 nmol/l >86%; 10 micromol/l >67%) was found. In contrast, subplasmalemmal Ca2+ response, which favors smooth muscle relaxation caused by activation of Ca2+-activated K+ channels, was reduced by 38% in diabetic patients as compared with control subjects, indicating a significant change in the subcellular Ca2+ distribution in vascular smooth muscle cells in diabetic patients. In contrast to the altered Ca2+ signaling found in freshly isolated cells from diabetic patients, in cultured smooth muscle cells isolated from control subjects and diabetic patients, no difference in the intracellular Ca2+ signaling to stimulation with either K+ or NE was found. Furthermore, production of superoxide anion (*O2-) in intact and endothelium-denuded arteries from diabetic patients was increased by 150 and 136%, respectively. Incubation of freshly isolated smooth muscle cells from control subjects with the *O2- -generating system xanthine oxidase/hypoxanthine mimicked the effect of diabetic patients on subcellular Ca2+ distribution in a superoxide dismutase-sensitive manner. We conclude that in diabetic subjects, smooth muscle reactivity is increased because of changes in subcellular Ca2+ distribution on cell activation. Increased *O2- production may play a crucial role in the alteration of smooth muscle function.

Multidrug resistance in acute leukemia: a comparison of different diagnostic methods.

Accurate measurement of P-glycoprotein (P-170) expression in clinical samples still remains a controversial issue. In this study tumor cell P-170 expression was assessed in 29 patients suffering from acute leukemia (17 acute myeloid leukemia (AML) and 12 acute lymphoblastic leukemia (ALL)) using three different techniques: flow cytometry measuring rhodamine 123 (Rh123) efflux (functional level), immunocytochemistry (protein level) and RT-PCR (mRNA level). Rh123 efflux was detectable in 10/29 (34%) of all cases, in 9/17 (53%) of AML and in 1/12 (8%) of ALL samples. In AML patients a significant association of CD34 expression and P-170 activity was observed (P < 0.02). All AML patients with the FAB subtype M5 were Rh123 negative (P < 0.007). Cytospin preparations were analyzed for staining with monoclonal antibodies JSB1 and MM4.17. Eight of 16 (50%) AML and 0/9 (0%) ALL cases expressed the multidrug resistance (MDR) protein assessed by JSB1. With MM4.17 87% of AML and 50% of ALL patients were scored positive. Agreement between both antibodies was found in only 13/23 (57%) samples. Extracted RNA from 12 patients was analyzed by RT-PCR to evaluate the expression of MDR1 and multidrug resistance-associated protein (MRP) mRNA. An increased level of MDR1 mRNA was detectable in 4/7 AML and 0/5 ALL cases. MRP expression was found in 3/7 AML and 0/5 ALL patients. Comparison of Rh123 assay and immunocytochemistry revealed a very good correlation when using MoAb JSB1 (P < 0.004) but not with MM4.17 (not significant (NS)). JSB1 also showed a much better association with the PCR results (P < 0.05) than MM4.17 (NS). Finally, we compared the results of the functional Rh123 assay and RT-PCR and observed a high correlation for Rh123/MDR1 (r = 0.819, P < 0.001) but low for Rh123/MRP (r = 0.562, NS). We conclude that measurement of Rh123 efflux and immunocytochemical staining of cytospin preparations with JSB1 allows the accurate monitoring of P-170 expression in acute leukemia. The simplicity of these two MDR assays suggests their use for routine MDR screening.

PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) P-glycoprotein by bryostatin 1.

Bryostatin 1 is a new antitumor agent which modulates the enzyme activity of protein kinase C (PKC, phospholipid-Ca2+-dependent ATP:protein transferase, EC 2.7.1.37). Several reports have suggested that the pumping activity of the multidrug resistance gene 1 (MDR1)-encoded multidrug transporter P-glycoprotein (PGP) is enhanced by a PKC-mediated phosphorylation. It was shown here that bryostatin 1 was a potent modulator of multidrug resistance in two cell lines over-expressing a mutant MDR1-encoded PGP, namely KB-C1 cells and HeLa cells transfected with an MDR1-V185 construct (HeLa-MDR1-V185) in which glycine at position 185 (G185) was substituted for valine (V185). Bryostatin 1 is not able to reverse the resistance of cells over-expressing the wild-type form (G185) of PGP, namely CCRF-ADR5000 cells and HeLa cells transfected with a MDR1-G185 construct (HeLa-MDR1-G185). Treatment of HeLa-MDR1-V185 cells with bryostatin 1 was accompanied by an increase in the intracellular accumulation of rhodamine 123, whereas no such effect could be observed in HeLa-MDR1-G185 cells. HeLa-MDR1-V185 cells expressed the PKC isoforms alpha, delta and zeta. Down-modulation of PKC alpha and delta by 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect the drug accumulation by bryostatin 1. Bryostatin 1 depleted PKC alpha completely and PKC delta partially. In HeLa-MDR1-V185 cells, short-term exposure to bryostatin 1, which led to a PKC activation, was as efficient in modulating the pumping activity of PGP as long-term exposure leading to PKC depletion. Bryostatin 1 competed with azidopine for binding to PGP in cells expressing the MDR1-V185 and MDR1-G185 forms of PGP. It is concluded that bryostatin 1: i) interacts with both the mutated MDR1-V185 and the wild-type MDR1-G185; ii) reverses multidrug resistance and inhibits drug efflux only in PGP-V185 mutants; and iii) that this effect is not due to an interference of PKC with PGP. For gene therapy, it is important to reverse the specific resistance of a mutant in the presence of a wild-type transporter and vice versa. Our results show that it is possible to reverse a specific mutant PGP.

Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities.

We investigated endocytosis activity, uptake of miltefosine (hexadecylphosphocholine), phospholipid and cholesterol content, the cell cycle, and apoptosis in 13 tumor cell lines (MCF7, MCF7/ADR, KB-3-1, KB-8-5, KB-C1, HeLa, HeLa-MDR1-G185, HeLa-MDR1-V185, CCRF/CEM, CCRF/VCR1000, CCRF/ADR5000, HL-60, HL-60/AR) with different sensitivities to treatment with the antitumor phospholipid analogues miltefosine and D-21266 (octadecyl-(N,N-dimethyl-piperidino-4-yl)-phosphate). In this panel of cell lines, MDR1 (multidrug resistance gene 1)- and MRP1 (multidrug resistance-associated protein)-expressing cells were found to be slightly more resistant to both compounds than sensitive parental cells. No correlation was found between resistance to miltefosine and endocytosis, intracellular concentration of miltefosine, the phospholipid and cholesterol content, induction of apoptosis, or cell cycle alterations in all the cell lines tested. Wild-type p53 containing WMN Burkitt's lymphoma cells and wild type p53-deficient CA46 exhibited similar sensitivities to miltefosine. The low percentage of apoptosis induced in MCF7 cells lacking caspase 3 indicated that caspase 3 seems to play an essential role in miltefosine-induced apoptosis.

Reversal of multidrug resistance by B859-35, a metabolite of B859-35, niguldipine, verapamil and nitrendipine.

It has been shown previously that verapamil and other calcium antagonists and calmodulin inhibitors can reverse multidrug resistance. We compared the potency of the dihydropyridine derivatives (4R)-3-[3-(4,4-diphenyl-1-piperadinyl)-propyl]-5-methyl-1,4-dihydr o-2,6- dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (B859-35), a metabolite of B859-35, niguldipine and (R)-nitrendipine to that of (RS)-verapamil in reversing multidrug resistance. The accumulation of the fluorescent dye rhodamine 123, which is transported by the P-glycoprotein, was determined by a flow cytometer. Multidrug-resistant human HeLa KB-8-5 and Walker rat carcinoma cells were incubated in the presence and in absence of the drugs indicated above. We found that 0.1 microM B859-35 increases the accumulation of rhodamine 123 in multidrug-resistant KB-8-5 and Walker cells more effectively than 1 microM (RS)-verapamil. In sensitive KB-3-1 cells addition of the drugs had no significant influence on the accumulation of rhodamine 123. IN KB-8-5 cells, 10 nM Adriamycin caused a reduction of cell growth to 85% compared to untreated controls (= 100%). If 1 microM B859-35, B859-35 metabolite, niguldipine, verapamil or (R)-nitrendipine was added to 10 nM Adriamycin, growth reduction compared with untreated controls increased to 12%, 11%, 23%, 63%, and 82% respectively. The effect of 0.1 microM B859-35 was a reduction in proliferation to 38%, that of 0.1 microM verapamil to 72%. These data illustrate that B859-35, a compound with antitumor activity in several tumors, is at least ten times more potent than racemic verapamil in reversing multidrug resistance.

Protein kinase C isoenzymes, p53, accumulation of rhodamine 123, glutathione-S-transferase, topoisomerase II and MRP in multidrug resistant cell lines.

We investigated whether the expression of protein kinase C (PKC) isoenzymes, topoisomerase II alpha, II beta, multidrug resistance associated protein (MRP), p53 or the activity of glutathione-S- transferase (GST) are additional factors contributing to the resistance mediated by multidrug resistance gene 1 (mdr 1). the cell lines employed for these studies were human lymphoblastoid CCRF cells selected for resistance with actinomycin D, vincristine and adriamycin, KB-3-1 and matched resistant KB-8-5 and KB-C1 cells (selected with colchicine), and a HeLa cell line, in which the resistance was obtained by transfection with the mdr1-gene. Analysis of PKC isozymes showed that there is no correlation of a specific isoenzyme with resistance, although minor differences in the expression were observed. In vincristine and adriamycin selected cells, topoisomerase II alpha- and II beta-MRNA levels were reduced, and in vincristine selected cells the MRP-mRNA was elevated compared with the sensitive line. In KB cells the levels of topoisomerase II alpha and II beta mRNA were increasing with the resistance. Expression of p53 did not correlate with Pgp levels. In summary, MRP and topoisomerase II may contribute to the mdr1 -mediated resistance in some cell lines, but PKC, p53 and GST seem to be of minor or no importance.

The involvement of protein kinase C isoenzymes alpha, epsilon and zeta in the sensitivity to antitumor treatment and apoptosis induction.

In order to obtain additional information on the involvement of protein kinase C (PKC) isoenzymes in the resistance of cells to anticancer drugs and in the induction of apoptosis, we employed antisense oligonucleotides to PKC alpha and PKC zeta, CGP 53506, a new inhibitor of PKC alpha, and cells overexpressing PKC alpha, PKC epsilon and PKC zeta. We found that in HeLa cells which express PKC alpha and zeta, down-modulation of either PKC alpha or PKC zeta with antisense oligonucleotides induced apoptosis. The PKC alpha selective inhibitor CGP 53506 reduced the proliferation rate of PKC alpha overexpressing NIH3T3 cells more than that of wild-type cells and induced apoptosis, indicating that such a PKC alpha inhibitor may be useful in the treatment of tumors overexpressing PKC alpha such as glioblastomas. NIH3T3 cells overexpressing PKC alpha were more resistant, whereas NIH3T3 cells overexpressing PKC epsilon or PKC zeta were more sensitive to treatment with cis-platin, adriamycin or gamma-irradiation compared to parental NIH3T3 wild-type cells. The observed resistance and sensitization corresponded to the extent of apoptosis induced by these treatments. Alterations in the expression of p53, bcl-2 and bax in the PKC isoenzyme overexpressing cells indicate that these proteins may be involved in the different sensitivities of these cells.

Dioxin residues in the edible tissue of finishing pigs after dioxin feeding.

The aim of this study was to determine the contamination of finishing pigs with polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) after feeding either uncontaminated feed or feed contaminated with 0.75, 2 or 4 ng/kg toxic equivalents (TEQ; calculated by multiplying individual congener concentrations by congener-specific toxicity equivalency factors). The feed was mixed with pure substances of PCDD/Fs to get the intended contamination. Five groups of six piglets each were fed contaminated feed, one group of five piglets served as control. One group was fed contaminated feed (4 ng TEQ/kg) only for the rearing period (6 weeks), and another group for the first 8 weeks of the fattening period (4 ng TEQ/kg feed). The other groups received the contaminated feed during the 12-weeks fattening period. After slaughtering, the edible parts of the belly, loin and fore-end were collected and homogenized. The samples of group 2 and 4a were investigated uncooked as well as roasted. Fattening yield and feed conversion (kg feed/kg weight gain) of the animals of all groups were in the normal range (final weight 109.7 kg; feed conversion 2.55-2.69 kg). The PCDD/F-content in 1 kg fat of the belly, loin and fore-end in relation to the intake was between 0.016 (4 ng TEQ/kg feed for a 6-weeks rearing period) and 1.39% (fore-end; 2 ng TEQ/kg feed for 12-weeks fattening period). There was a decrease in dioxin residues after a 12-weeks period but not after a 4-weeks period of feeding an uncontaminated feed. When feed contaminated with 0.75, 2 and 4 ng TEQ/kg was given for a 12-weeks fattening period, the residue concentrations of PCDD/F-TEQ in 1 kg belly was 0.455, 1.07 and 1.55 ng, in 1 kg fore-end 0.04 ng, 0.32 ng and 0.34 ng and in 1 kg loin 0.015 ng, 0.07 ng and 0.30 ng respectively. Roasting had no influence on the dioxin-residues. The residues per g belly fat exceed the maximum limits for dioxin in food of 0.6 pg WHO-PCDD/F-TEQ/g fat (EC Recommendations 2002/201/EC), when feed containing 0.75 ng PCDD/F-TEQ/kg is given for 12 weeks. When feed containing 0.4 ng TEQ (maximum content; EC recommendations 2002/201/EC) is given for 12 weeks, approximately 0.55 pg TEQ/g fat can be expected in the food. This value is within the action level of 0.6 pg/g fat of porks. In conclusion, the results of the study allow prediction of dioxin residues in the edible tissue of pork, if the feed contamination is known and the amount of feed intake can be estimated.

Vascular targets of redox signalling in diabetes mellitus.

There is overwhelming evidence for an involvement of reactive oxygen species (ROS) in the pathogenesis of diabetes-associated vascular complications. However, neither the exact source of the ROS initiating cascades leading to cell dysfunction in diabetes nor their chemical nature is fully understood. Furthermore, despite our knowledge of the crucial role of ROS in diabetes, little is known about the actual targets and the molecular consequences of the interaction of ROS with cellular signalling pathways. Therefore, we aim to provide an overview of ROS (i.e. O2(*-), NO*, ONOO- and H2O2) and their vascular sources in diabetes and to summarise recent knowledge on the mechanisms underlying increased ROS production within the vascular wall. In addition, possible targets of diabetes and ROS within the vasculature are discussed. These include, the effects of ROS on small guanine nucleotide binding proteins, the cytoskeleton, protein kinases (e.g tyrosine kinases), metalloproteinases, ion homeostasis and transcriptional regulation. Such analysis makes it clear that the generation of ROS could affect a large number of various signalling pathways and proteins. Thus, a better knowledge of the functional diversity and pathological consequences of each individual pathway activated by ROS id essential to understand the mechanisms of diabetes-associated vascular complications.

Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota.

Atypical protein kinase C-iota (aPKCiota) plays an important role in mitogenic signaling, actin cytoskeleton organization, and cell survival. Apart from the differences in the regulatory domain, the catalytic domain of aPKCiota differs considerably from other known kinases, because it contains a modification within the glycine-rich loop motif (GXGXXG) that is found in the nucleotide-binding fold of virtually all nucleotide-binding proteins including PKCs, Ras, adenylate kinase, and the mitochondrial F1-ATPase. We have used site-directed mutagenesis and kinetic analysis to investigate whether these sequence differences affect the nucleotide binding properties and catalytic activity of aPKCiota. When lysine 274, a residue essential for ATP binding and activity conserved in most protein kinases, was replaced by arginine (K274R mutant), aPKCiota retained its normal kinase activity. This is in sharp contrast to results published for any other PKC or even distantly related kinases like phosphoinositide 3-kinase gamma, where the same mutation completely abrogated the kinase activity. Furthermore, the sensitivity of aPKCiota for inhibition by GF109203X, a substance acting on the ATP-binding site, was not altered in the K274R mutant. In contrast, replacement of Lys-274 by tryptophan (K274W) completely abolished the kinase activity of PKCiota. In accordance with results obtained with other kinase-defective PKC mutants, in cultured cells aPKCiota-K274W acted in a dominant negative fashion on signal transduction pathways involving endogenous aPKCiota, whereas the effect of the catalytically active K274R mutant was identical to the wild type enzyme. In summary, aPKCiota differs from classical and novel PKCs also in the catalytic domain. This information could be of significant value for the development of specific inhibitors of aPKCiota as a key factor in central signaling pathways.

Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase.

Using single cell microfluorometry to monitor changes in bulk Ca2+ concentration ([Ca2+]bulk) and the whole-cell configuration of the patch clamp technique to measure K+ currents (voltage clamp) and membrane potential (current clamp), the mechanisms of histamine-induced Ca2+ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. In single cells, histamine (10 microM) evoked sinusoidal Ca2+ oscillations in low extracellular Ca2+ concentrations ([Ca2+]o = 10-30 microM). In contrast, histamine did not initiate Ca2+ oscillations either in the absence of extracellular Ca2+ (10 microM EGTA) or in the presence of 2.5 mM extracellular Ca2+. Ca2+ oscillations were accompanied by rhythmic activation of Ca2+-activated K+ (KCa) channels and membrane hyperpolarization of 18.1 +/- 3.9 mV. Hence, cell depolarization with 70 mM extracellular K+ or the inhibition of non-selective cation channels (NSCCs) and KCa channels by 10 microM Loe 908 and 10 mM tetrabutylammonium prevented histamine-evoked Ca2+ oscillations. Preventing Na+-Ca2+ exchange (NCX) by 10 microM 2', 4'-dichlorobenzamil, or removal of extracellular Na+, abolished histamine-induced Ca2+ oscillations. Lowering the extracellular Na+ concentration and thus promoting the reversed mode of NCX (3Na+ out and 1Ca2+ in) increased the amplitude and frequency of histamine-induced Ca2+ oscillations by 25 and 13 %, respectively. Hence, in the absence of extracellular Ca2+, 10 microM histamine induced an elevation of intracellular Na+ concentration in certain subplasmalemmal domains. The inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2,5-di-tert-butyl-1, 4-benzo-hydroquinone (15 microM) prevented histamine-induced Ca2+ oscillations. In addition, blockage of ryanodine-sensitive Ca2+ release (RsCR) by 25 microM ryanodine blunted Ca2+ oscillations. In endothelial cells that were treated for 16 h with 10 microM nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induced Ca2+ oscillations were found. We conclude that in low [Ca2+]o conditions histamine-induced Ca2+ oscillations depend on transmembrane Na+ loading through NSCCs that leads to Ca2+ entry via NCX. Cation influx is controlled by KCa channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca2+ entering needs to be sequestrated via SERCA into sER to become released by RsCR to evoke Ca2+ spiking. These data further support our previous work on localized Ca2+ signalling as a key phenomenon in endothelial Ca2+ homeostasis.

Complex formation and cooperation of protein kinase C theta and Akt1/protein kinase B alpha in the NF-kappa B transactivation cascade in Jurkat T cells.

Protein kinase C theta (PKC theta) is known to induce NF-kappa B, an essential transcriptional element in T cell receptor/CD28-mediated interleukin-2 production but also T cell survival. Here we provide evidence that PKC theta is physically and functionally coupled to Akt1 in this signaling pathway. First, T cell receptor/CD3 ligation was sufficient to induce activation as well as plasma membrane recruitment of PKC theta. Second, PKC theta selectively cooperated with Akt1, known to act downstream of CD28 co-receptor signaling, in activating a NF-kappa B reporter in T cells. Third, Akt1 function was shown to be required for PKC theta-mediated NF-kappa B transactivation. Fourth, PKC theta co-immunoprecipitated with Akt1; however, neither Akt1 nor PKC theta served as a prominent substrate for each other in vitro as well as in intact T cells. Finally, plasma membrane targeting of PKC theta and Akt1 exerted synergistic transactivation of the I-kappa B kinase beta/inhibitor of NF-kappa B/NF-kappa B signaling cascade independent of T cell activation. Taken together, these findings suggest a direct cross-talk between PKC theta and Akt1 in Jurkat T cells.

Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123.

Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P-glycoprotein (P-gp), we developed a functional flow cytometric assay for the detection of multidrug-resistant (MDR) cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established that enabled demonstration of significant differences in Rh123 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two-colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rh123 was associated with P-gp expression, the leukaemic cells were stained with the monoclonal antibody MRK-16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P-gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two-colour flow cytometry was much easier to perform with Rh123 than with daunorubicin. Our results indicate that flow cytometric measurement of Rh123 accumulation/efflux proves applicable to detect MDR cells in heterogenous clinical samples.

Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440).

The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound. Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.

Reversal of multidrug resistance by the staurosporine derivatives CGP 41251 and CGP 42700.

It has been shown previously that the staurosporine derivative CGP 41251, a specific inhibitor of protein kinase C (IC50 = 50 nM), exhibits antitumor activity and reverses mdr1 mediated multidrug resistance. At present, the compound is evaluated as an anticancer drug in clinical phase I trials. We compared the effects of CGP 41251 with CGP 42700, another staurosporine derivative, which exhibits low protein kinase C inhibiting activity (IC50 = > 100 microM). We found that in contrast to CGP 41251, CGP 42700 does not show antiproliferative activity in HeLa and KB cells in tissue culture (up to a concentration of 10 microM). We compared both compounds for their ability to reverse mdr1-mediated resistance in KB-C1 and in HeLa-MDR1 cells (transfected with the mdr1 gene). CGP 42700 is able to reverse mdr1-mediated resistance to a similar extent as CGP 41251. The intracellular accumulation of rhodamine 123 in KB-C1 cells following pretreatment with CGP 41251 for 30 min was higher than that following treatment with CGP 42700 if determined in medium without serum. However, quantitation of rhodamine efflux in an ex vivo assay using human CD8+ cells in serum showed that CGP 42700 is more effective in inhibiting the efflux of rhodamine 123 than CGP 41251. We conclude from our results that (1) CGP 42700 is more effective in reversal of multidrug resistance in serum than CGP 41251, indicating that the compound may be useful for treatment of patients, and (2) CGP 42700 does not inhibit protein kinase C and cell proliferation and, therefore, may be less toxic and elicit less side effects in humans than other chemosensitizers.

Intercellular signalling within vascular cells under high D-glucose involves free radical-triggered tyrosine kinase activation.

AIMS/HYPOTHESIS: Diabetes mellitus is associated with endothelial dysfunction in human arteries due to the release of superoxide anions (*O(2)(-)) that was found to occur predominantly in smooth muscle cells (SMC). This study was designed to elucidate the impact of high glucose concentration mediated radical production in SMC on EC. Pre-treatment of vascular SMC with increased D-glucose enhanced release of *O(2)(-). METHODS: Microscope-based analyses of intracellular free Ca(2+) concentration (fura-2), immunohistochemistry (f-actin) and tyrosine kinase activity were performed. Furthermore, RT-PCR and Western blots were carried out. RESULTS: Interaction of EC with SMC pre-exposed to high glucose concentration yielded changes in endothelial Ca(2+) signalling and polymerization of f-actin in a concentration-dependent and superoxide dismutase (SOD) sensitive manner. This interaction activated endothelial tyrosine kinase(s) but not NFkappaB and AP-1, while SOD prevented tyrosine kinase stimulation but facilitated NFkappaB and AP-1 activation. Erbstatin, herbimycin A and the src family specific kinase inhibitor PP-1 but not the protein kinase C inhibitor GF109203X prevented changes in endothelial Ca(2+) signalling and cytoskeleton organization induced by pre-exposure of SMC to high glucose concentration. Adenovirus-mediated expression of kinase-inactive c-src blunted the effect of pre-exposure of SMC to high glucose concentration on EC. CONCLUSIONS/INTERPRETATION: These data suggest that SMC-derived *O(2)(-) alter endothelial cytoskeleton organization and Ca(2+) signalling via activation of c-src. The activation of c-src by SMC-derived radicals is a new concept of the mechanisms underlying vascular dysfunction in diabetes.

Regulation of phospholipase D isoenzymes by transforming Ras and atypical protein kinase C-iota.

The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-iota with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-iota, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-iota, whereas PLD2 was less effective in this respect. The data suggest that PKC-iota acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-iota. The data are discussed as indicating a putative signalling cascade comprising Ras-->PLD1b-->PKC-iota-->PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.