A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 31-31
Author(s):  
Jin-Qiu Chen ◽  
Madeleine Heldman ◽  
Michelle Herrmann ◽  
Noemi Kedei ◽  
Peter Blumberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Relative Abundance ◽  
U937 Cells ◽  
Bryostatin 1 ◽  
Absolute Quantitation ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

31 Background: The human prostate cell line LNCaP and the human myelocytic leukemia cell line U937 differ dramatically in their responses to the two protein kinase C (PKC) targeted ligands phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 and show complex differences in the patterns of transcriptional responses that they induce. Quantitation of relative abundance of individual PKC isoforms in the two cell lines may help to link the downstream effects of the two compounds to these isoforms. Methods: Simple Western is a capillary-based automated Western system recently developed by ProteinSimple. All steps following sample preparation are fully automated in the Simple Western system, including sample loading, size-based protein separation, immunoprobing, washing, detection and data analysis. Simple Western is gel-free and blot-free, uses less amount of samples, and produces highly quantitative, reproducible information that cannot be generated using regular Western assays. Using the Simple Western system, we developed a method for absolute quantitation of endogenous proteins in cell lysates and quantified PKC isoforms in LNCaP and U937 cells. Results: PKC isoforms were measured at levels of picogram or sub-picogram per nanogram cell lysate. PKC delta was identified as the dominant PKC isoforms in both cell lines. In LNCaP cells, PKC delta expression is ~20-fold higher than PKC alpha, ~40-fold higher than PKC epsilon, and at least 20-fold higher than PKC beta. In U937 cells, PKC delta expression is similar to PKC beta, at least 200 fold higher than PKC alpha, and ~50-fold higher than PKC epsilon. Conclusions: The Simple Western system, with its high-quality data quantitation and excellent assay reproducibility, allowed us to detect both the relative abundance of the PKC isoforms and their absolute quantitation in the tested cells. It circumvents the problem that antibodies of different affinities for different proteins yield a misleading impression of relative abundance and it provides an approach to accurately correlate protein quantities with their function.

Protein kinase C-epsilon is the likely mediator of mucin exocytosis in human colonic cell lines

1997 ◽  
Vol 272 (1) ◽  
pp. G31-G37 ◽  
Author(s):  
D. H. Hong ◽  
J. F. Forstner ◽  
G. G. Forstner
Keyword(s):  
Protein Kinase C ◽  
Cell Line ◽  
Cell Lines ◽  
Mucin Secretion ◽  
T84 Cells ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha ◽  
Colonic Cells ◽  
Ht 29

The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.

Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion

1997 ◽  
Vol 272 (1) ◽  
pp. C263-C269 ◽  
Author(s):  
D. Zoukhri ◽  
R. R. Hodges ◽  
C. Sergheraert ◽  
A. Toker ◽  
D. A. Dartt
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Secretion ◽  
Phorbol Ester ◽  
Lacrimal Gland ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha ◽  
Kinase A

In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.

Do adult rat ventricular myocytes express protein kinase C-alpha?

1997 ◽  
Vol 272 (5) ◽  
pp. H2485-H2491 ◽  
Author(s):  
V. Rybin ◽  
S. F. Steinberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Chromatographic Purification ◽  
Adult Rat ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Although calcium-insensitive protein kinase C (PKC) isoforms (PKC-epsilon and PKC-delta) are consistently detected in adult ventricular myocytes, the evidence that adult ventricular myocytes also express calcium-sensitive PKC-alpha is inconsistent. The current study used four different anti-PKC-alpha-antibodies to resolve some of the uncertainties regarding the immunodetection of PKC-alpha in adult ventricular myocytes. Three of the antibodies used in this study barely (GIBCO-BRL) or rather faintly (Transduction Laboratories and Seikagaku America) recognize PKC-alpha in crude preparations from adult ventricular myocytes. Although each of these antibodies recognizes a prominent 80-kDa band, which is similar in size to PKC-alpha, this represents nonspecific immunoreactivity and should not be confused with PKC-alpha. This conclusion is based on peptide-blocking experiments (GIBCO-BRL), the absence of the requisite sensitivity to calcium- and phorbol 12-myristate 13-acetate-induced translocation (Seikagaku America and Transduction Laboratories), and/or the failure to copurify with PKC-alpha on DEAE-Sephacel chromatography. Nevertheless, an antibody from Upstate Biotechnology clearly recognizes PKC-alpha and not other unrelated nonspecific immunoreactive species in crude preparations from adult ventricular myocytes. Each of the antisera used in this study could detect PKC-alpha immunoreactivity following chromatographic purification of the samples to enrich for PKC-alpha and remove nonspecific immunoreactive proteins. These results suggest that PKC-alpha is expressed by adult ventricular myocytes and argue that differences in the sensitivity and/or specificity of available antisera contribute to at least some of the confusion regarding PKC-alpha expression in adult ventricular myocytes.

Propofol-induced Activation of Protein Kinase C Isoforms in Adult Rat Ventricular Myocytes

2006 ◽  
Vol 104 (5) ◽  
pp. 970-977 ◽  
Author(s):  
Peter J. Wickley ◽  
Xueqin Ding ◽  
Paul A. Murray ◽  
Derek S. Damron
Keyword(s):  
Ventricular Myocytes ◽  
Intracellular Location ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Pkc Isoforms ◽  
Pkc Alpha ◽  
The Individual

Background Myocardial protection by anesthetics is known to involve activation of protein kinase C (PKC). The authors' objective was to identify the PKC isoforms activated by propofol in rat ventricular myocytes. They also assessed the intracellular location of individual PKC isoforms before and after treatment with propofol. Methods Freshly isolated ventricular myocytes were obtained from adult rat hearts. Immunoblot analysis of cardiomyocyte subcellular fractions was used to assess translocation of individual PKC isoforms before and after exposure to propofol. An enzyme-linked immunosorbent assay kit was used for measuring PKC activity. Immunocytochemistry and confocal microscopy were used to visualize the intracellular location of the individual PKC isoforms. Results Under baseline conditions, PKC-alpha, PKC-delta, and PKC-zeta were associated with both the cytosolic and membrane fractions, whereas PKC-epsilon was exclusively located in the cytosolic fraction. Propofol (10 microM) caused translocation of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from cytosolic to membrane fraction and increased total PKC activity (211 +/- 17% of baseline; P = 0.003) in a dose-dependent manner. Immunocytochemical localization of the individual PKC isoforms demonstrated that propofol caused translocation of PKC-alpha to the intercalated discs and z-lines; PKC-delta to the perinuclear region; PKC-epsilon to sites associated with the z-lines, intercalated discs, and the sarcolemma; and PKC-zeta to the nucleus. Conclusions These results demonstrate that propofol causes an increase in PKC activity in rat ventricular myocytes. Propofol stimulates translocation of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to distinct intracellular sites in cardiomyocytes. This may be a fundamentally important cellular mechanism of anesthesia-induced myocardial protection in the setting of ischemia-reperfusion injury.

Expression Profiles of VDR-Induced Differentiation of atRA Refractory AML Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3291-3291
Author(s):  
Aurelie Baudet ◽  
Ronan Quere ◽  
Alice Roman ◽  
Jacques Marti ◽  
Therese Commes
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Growth Arrest ◽  
Promyelocytic Leukemia ◽  
S Phase ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Induced Differentiation ◽  
Acute Leukemias

Abstract Whatever the success of all-trans retinoic acid (atRA) therapy in acute promyelocytic leukemia (FAB AML3), other AML are poorly responsive to atRA and don’t benefit of any differentiation therapy. Moreover, primarily responsive patients often acquire resistance. Among molecules that control normal myelopoiesis, 1,25(OH)2-vitaminD3 (VD3), through its receptor VDR, allows maturation of myelomonocytic precursors. If VD3 has low effects on AML cells differentiation, its activity can be enhanced by various molecules. So, we tested several differentiation activators in combination with the non calcemiant VDR agonist EB1089 (EB) (Leo Pharmaceutics) on two atRA-refractory cell lines. Thus, U937 cells reproduce myelomonocytic acute leukemias that are strongly sensitive to VDR-induced differentiation and with a poor atRA sensitivity. Parallel, NB4-derived LR2 model mimics promyelocytic leukemia with acquired atRA resistance and has lower response to VD3. First, we confirm VDR-sensitivity of those cell lines. Moreover, as expected, U937 cells respond to EB1089 at lower dose (1nM) than NB4-LR2 (10nM) as showed by CD11b myeloid marker level. Among tested activators, the cytokine TGFb induces the most complete differentiation for both cell lines. After a 72 hour-treatment, only 5% of cells are still clustered in S phase. In addition, all are positive for the monocyte marker CD14 which expression reaches to 10 and 6 time fold the base level for U937 and NB4-LR2 respectively. At our knowledge, it is the first model of complete differentiation of NB4-LR2. Even if this agent can’t be used in therapy, it provides a reference for comparing other potential differentiating molecules. In fact, Histones Deacetylases Inhibitors (HDI) and arsenate (ATO) both benefit to VDR-induced differentiation of atRA refractory cell lines. Specifically, ATO benefits to EB-induced differentiation, as both U937 and NB4-LR2 cells acquire monocytic phenotype (CD14 expression) even though growth arrest is weaker. Indeed there are still 10–15% of S-phase clustered cells. Concerning the association of HDI valproic acid (VPA) and trichostatine A (TSA) with EB, growth arrest is strongest for both cell lines (5–10% S phase clustered cells) at 1mM and 100nM respectively. For phenotype markers, responses vary: U937 cells only respond to TSA combination whereas NB4-LR2 cells differentiate with TSA or VPA. Moreover, only TSA-EB treated cells become positive for CD14 even if expression is low. The addition of LGD1069, agonist of the Retinoid X receptor, a obligatory partner of VDR, improves HDI effects in all measured parameters. In case of NB4-LR2 cell line, flow cytometry data were reinforced by expression profile analyses. A hundred messengers were selected from bibliography and SAGE libraries of the promyelocytic cell line NB4 (unpublished data) and profiles were established by semi-quantitative real time PCR on low density array. Manual clusterization after comparison to granulocytic and monocytic differentiation (atRA-treated NB4 and VDTGFb-treated NB4-LR2 cells respectively) allows selection of about 10 messengers sufficient to predict patients’ response and to specify differentiation. In addition, the clinical use of VDR agonist, HDI and ATO is conceivable because of low side effects.

Effects of rmhTRAIL Combined with Daunorubicin in Inducing Apoptosis of Leukemia Cell Lines and Its Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1986-1986
Author(s):  
Xuejun Zhang ◽  
Li Wen ◽  
Fuxu Wang ◽  
Ling Pan ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mrna Level ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Expression Level ◽  
U937 Cells ◽  
Leukemia Cell Lines ◽  
Before And After

Abstract Tumor Necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) is a new member of TNF superfamily discovered recently. Several studies showed that TRAIL can preferentially induce apoptosis in a variety of tumor cells, while most normal cells tested do not appear to be sensitive to TRAIL. In the present study, we treated K562 and U937 leukemia cell lines with recombinant mutant human TRAIL (rmhTRAIL) alone or together with daunorubicin (DNR) to investigate the apoptosis of the treated cells and the synergistic reaction of rmhTRAIL and DNR. The normal cell line MRC-5 was used as control. The expression of four TRAIL receptors mRNA (death receptor DR4 and DR5, decoy receptor DcR1 and DcR2) in the cells lines were detected before and after the treatment by DNR. (1) AO-EB double staining and TUNEL staining were used to evaluate the morphological change of leukemia cell lines before and after the treatment. The results showed that rmhTRAIL could induce the apoptosis of leukemia cell lines and a dose-dependent manner was found in leukemia cell lines but not in MRC-5 cell lines. (2) The growth inhibition rate of leukemia cell lines induced by rmhTRAIL alone or combined with DNR was examined with MTT assays. Different concentrations of rmhTRAIL(8, 40, 200, 1000ng/mL)alone or combined with DNR(8, 40, 200, 1000ng/mL) was used. The result showed a dose-dependent growth inhibition by rmhTRAIL alone for K562- and U937-cell line (P<0.05) also, but not for MRC-5 cell line (P>0.05). The IC50 for K562 cells and for U937 cells had no statistic difference (538.80 vs 301.56ng/mL, P>0.05). In leukemia cell lines, the growth inhibition rates in combination groups were much higher than in rmhTRAIL or DNR alone groups (P<0.05), and no synergistic killing effects was found in MRC-5 cells (P<0.05). It was concluded that rmhTRAIL had synergistic effects with DNR in the growth inhibition of K562 and U937 cells. (3). To explore the antitumor mechanisms of rmhTRAIL combined with DNR, the expression level of the DR4, DR5 and DcR1, DcR2 mRNA in these three cell lines was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and after the treatment with DNR. The high expression of DR4,DR5 mRNA in the tested cells were observed before the treatment of DNR, while very low or even undetectable expression level of DcR1 and DcR2 mRNA were observed in U937 and K562 cells, and a high expression level of DcR1 and DcR2 mRNA in MRC-5 cells were observed. After 24 hours treatment of three cell lines with DNR (200ng/ml), the expression level of DR5 mRNA increased in K562 and U937 cells (P<0.05). DR4 mRNA also increased in K562 cells but not in U937 cells. There was no change in DcR1 and DcR2 mRNA level in three cell lines. The four receptors’ mRNA level in MRC-5 cells was not influenced by DNR. Our results indicated that rmhTRAIL could induce the apoptosis of leukemia cell lines, and DNR could enhance significantly the sensitivity of K562 and U937 cells to apoptosis induced by rmhTRAIL through up-regulation of death receptors. Therefore, we presumed TRAIL might be act as a new agent for biological therapy in leukemia.

scholarly journals Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney

2019 ◽  
Vol 53 (3) ◽  
pp. 154-164 ◽  
Author(s):  
Somchit Eiam-Ong ◽  
Mookda Chaipipat ◽  
Krissanapong Manotham ◽  
Somchai Eiam-Ong
Keyword(s):  
Rat Kidney ◽  
Kidney Cortex ◽  
Protein Abundance ◽  
Protein Levels ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Male Wistar Rats ◽  
Pkc Alpha

AbstractObjectives. Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney.Methods. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively.Results. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules.Conclusions. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.

Phorbol ester-stimulated exocytosis in lacrimal gland: PKC might not be the sole effector

1993 ◽  
Vol 264 (4) ◽  
pp. C1045-C1050 ◽  
Author(s):  
D. Zoukhri ◽  
C. Sergheraert ◽  
B. Rossignol
Keyword(s):  
Phorbol Ester ◽  
Lacrimal Gland ◽  
Inhibitory Effect ◽  
Complete Inhibition ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Diethylaminoethyl Cellulose ◽  
Half Maximal Effective Concentration ◽  
Pkc Alpha

In this work we show that, although both phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PdBu) stimulate the protein discharge in the rat lacrimal gland with the same half-maximal effective concentration (EC50 approximately 2 x 10(-7) M), PdBu is more efficient in eliciting this response compared with PMA. We also show that sphingosine and chelerythrine have no inhibitory effect on the protein discharge stimulated by PMA or PdBu at concentrations up to 2 x 10(-4) and 3 x 10(-5) M, respectively. With staurosporine, a complete inhibition could not be obtained even at 1 microM. However, only with trifluoperazine (TFP) we obtained a complete inhibition of the PMA-induced protein discharge at 10(-4) M TFP. On the other hand, we show that three diacylglycerol-permeant analogues (1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-didecanoyl-sn-glycerol) do not stimulate protein discharge. In a previous report from our laboratory (30), we showed that the rat lacrimal gland expresses the alpha-isoform of protein kinase C (PKC). In this study, using specific antibodies directed against the newly identified isoforms of PKC, we show on a diethylaminoethyl-cellulose fraction that, besides PKC-alpha, the rat lacrimal gland expresses PKC-epsilon, as previously suggested by Dartt et al. (11), and PKC-delta. Our results question the direct implication of PKC activity as a sole effector of the phorbol ester-stimulated protein secretion in the rat lacrimal gland.

Modulation Of The Glycolytic Metabolism In Acute Myeloid Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5045-5045
Author(s):  
Simone Mirabilii ◽  
Maria Rosaria Ricciardi ◽  
Matteo Allegretti ◽  
Roberto Licchetta ◽  
Martina Vincenzi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
U937 Cell ◽  
Fold Increase ◽  
Mek Inhibitor ◽  
U937 Cells ◽  
Apoptotic Effects ◽  
Glycolytic Rate ◽  
Acute Myeloid

Abstract Glycolysis is the central axis of cellular metabolism. The cancer cell bioenergetic status heavily relies on high glycolytic rates, even in aerobic conditions, thus sustaining the expensive processes of cell growth and proliferation. Growing evidences show that signaling aberrations - especially those involving PI3K/Akt/mTOR, HIF1a, Ras/Raf/MEK/ERK - are strictly connected to the establishment of a pro-glycolytic metabolism, through a multi-level crosstalk between proteins and metabolites that contribute to the acquisition of an energetic background granting a proliferative advantage. Here we investigated the glycolytic rate of resting and activated normal peripheral blood lymphocytes (NPBLs) and of acute myeloid leukemia (AML) cell lines. In an attempt to modulate the cellular metabolism for therapeutic intervention, we tested the following compounds that directly interfere with major metabolic or signaling pathways: dichloroacetate (DCA), a glycolysis inhibitor; aminooxyacetate (AOA), a glutaminolysis inhibitor; ST1326 (kindly given by Sigma-Tau), a fatty acid oxidation (FAO) inhibitor; and the MEK inhibitor PD0325901 (Selleck Chemicals). The cytotoxic drug effects were evaluated on two human leukemia cell lines, U937 and OCI-AML3, characterized by PI3K/Akt/mTOR and Ras/Raf/MEK/ERK hyperactivation, respectively. Cell counts, apoptosis (AnnexinV), glucose and lactate levels (GEM4000, Instrumentation Laboratory, UK) were measured. The glucose consumption rate (GCR) and lactate production rate (LPR) were calculated according to Li et al. (Biotechol. Appl. Biochem., 2005, 42, 73-80). Resting NPBLs were characterized by a very low glycolytic rate, according to their quiescent state, while cultured phytohemagglutinin-activated NPBLs displayed a remarkable increase in glycolytic rate: the GCR calculated over 72 hours showed a 25 fold-increase, while LPR had a of 10 fold-increase. Acute myeloid cell lines showed a high glucose catabolism: at 24h the U937 cell line, compared to activated NPBLs, had a 6.7 fold higher GCR, while the OCI-AML3 cell line showed a 4-fold increase. DCA exposure showed at 24h no detectable effect on GCR, LPR and apoptosis at concentrations ranging from 0.01 to 0.5mM on the U937 cell line. Apoptosis effects were detected only at higher concentrations: AnnexinV positive cells increased from 4.3 ± 1.5 (control) to 62.8 ± 16.4 (5mM) and 88.1 ± 16.8 (10mM). Exposure to AOA (24h at 1000µM) slightly increased GCR (1.23-fold) and LPR (1.22-fold) on U937 cells, followed by apoptotic effects at 72h: from 6.24 ± 4.2 (control) to 10.4 ± 0.8 at 100µM to 83.5 ± 0.7 at 1000µM. The FAO inhibitor ST1326 (10µM at 24h) induced a 3-fold increase of GCR in the U937 cell line. Apoptotic effects were seen in the U937 cells at 72h, from 5.0 ± 2.3 (control) to 35.9 ± 5.7 at 5µM to 64.1 ± 20.5 at 10µM. Conversely, GCR, LPR and apoptosis did not change on the OCI-AML3 line following ST1326 exposure. The MEK inhibitor PD0325901 caused a reduction of GCR and LPR on OCI-AML3 cells (6-fold GCR decrease, 2-fold LPR decrease at 100nM); apoptosis at 72h ranged from 6.3 ± 1.1 (control) to 15.5 ± 3.9 at 10nM to 45.3 ± 0.1 at 100nM. The U937 cells proved resistant to this compound, showing no metabolic perturbation and absence of apoptotic effects. In summary, this study indicates that exploiting the metabolism as a target for therapeutic intervention appears to be a promising new strategy. In fact, the inhibition of glycolysis by blocking either the activity of the enzymes that directly participate to the metabolic pathway or key components of cell signaling has proven to be effective in inducing apoptosis in AML cells. Interestingly, the opposing response to the various compounds observed in the two AML models may likely reflect their divergent signaling network, prompting further studies to evaluate the correlation between aberrant signal transduction pathways and peculiar metabolic profiles. Disclosures: Nicolai: Sigma Tau Pharmaceuticals: Employment.

scholarly journals Secretion of interleukin-8 by human-derived cell lines infected withMycobacterium bovis

2004 ◽  
Vol 13 (1) ◽  
pp. 45-49 ◽  
Author(s):  
Patricia Méndez-Samperio ◽  
Janet Palma-Barrios ◽  
Abraham Vázquez-Hernández ◽  
Elizabeth García-Martínez
Keyword(s):  
Infected Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Immunosorbent Assay ◽  
Calcium Influx ◽  
Enzyme Linked Immunosorbent Assay ◽  
U937 Cells ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Monocytic Cell

Background: The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovisBCG) in protecting humans against tuberculosis has prompted a search for the mechanisms through which BCG induces chemokines. In this study, our experiments were designed to determine the role of the transcription factor nuclear factor-κB (NF-κB) and intracellular calcium in the production of interleukin (IL)-8, a main chemotactic factor, by human-derived monocytic cell line U937 and by a human epithelial HEp-2 cell line infected withM.bovisBCG.Methods: The concentrations of IL-8 in culture supernatants of U937 cells or HEp-2 cells infected withM. bovisBCG were determined by enzyme-linked immunosorbent assay. We used sulfasalazine and curcumin, which are well-described inhibitors of NF-κB activity, and we used ethylenediamine tetraacetic acid to deplete extracellular Ca2+or used the cell-permeable agent 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester to chelate releasable intracellular stores of Ca2+in order to investigate the mechanisms through whichM.bovisBCG induces IL-8 secretion in our system.Results: The enzyme-linked immunosorbent assay showed that IL-8 protein secretion was elevated inM.bovis-infected cell lines. This effect was statistically significant(p<0.01). When calcium influx was suppressed inM.bovis-infected cell lines, IL-8 secretion was inhibited. Notably, specific inhibitors of NF-κB (sulfasalazine and curcumin) inhibitedM.bovis-induced IL-8 secretion from U937 cells or HEp-2 cells.Conclusions: Collectively, these results indicate that activation of NF-κB is an important signal transduction pathway inM.bovis-induced IL-8 secretion in monocytic or epithelial cells. Furthermore, the results showed that calcium influx had a direct effect on IL-8 secretion in U937 cells or HEp-2 cells infected withM.bovis.

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