Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer effects and pharmacological properties.

BACKGROUND AND PURPOSE: ABC multidrug transporters (MDR-ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti-cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first-generation, small-molecule, tyrosine kinase inhibitors (TKIs), including the Bcr-Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second-generation inhibitors of the Bcr-Abl tyrosine kinase activity. EXPERIMENTAL APPROACH: MDR-ABC transporter function was screened in both membrane- and cell-based (K562 cells) systems. Cytotoxicity measurements in Bcr-Abl-positive model cells were coupled with direct determination of intracellular TKI concentrations by high-pressure liquid chromatography-mass spectrometry and analysis of the pattern of Bcr-Abl phosphorylation. Transporter function in membranes was assessed by ATPase activity. KEY RESULTS: Nilotinib and dasatinib were high-affinity substrates of ABCG2, and this protein mediated an effective resistance in cancer cells against these compounds. Nilotinib and dasatinib also interacted with ABCB1, but this transporter provided resistance only against dasatinib. Neither ABCB1 nor ABCG2 induced resistance to bosutinib. At relatively higher concentrations, however, each TKI inhibited both transporters. CONCLUSIONS AND IMPLICATIONS: A combination of in vitro assays may provide valuable preclinical information for the applicability of novel targeted anti-cancer TKIs, even in multidrug-resistant cancer. The pattern of MDR-ABC transporter-TKI interactions may also help to understand the general pharmacokinetics and toxicities of new TKIs.

Reduction of Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation of ERK.

The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to abnormal transduction of growth and survival signals leading to chronic myeloid leukemia (CML). According to our previous observations, in vitro differentiation of several erythroid cell lines is accompanied by the downregulation of extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase (MAPK) activities. In this work we investigated whether ERKs have a decisive role in either the erythroid differentiation process or apoptosis of bcr-abl+ K562 cells by means of direct (MEK1/2 inhibitor UO126) and indirect (reduced Bcr-Abl function) inhibition of their activities. We found that both Gleevec and UO126 induced hemoglobin expression. Gleevec treatment reduced the phosphorylation of Bcr-Abl, ERK and STAT-5 for up to 24 h, decreased Bcl-XL levels, and induced caspase-3-dependent apoptosis. In contrast, UO126 treatment resulted in only a transient decrease of ERK activity and did not induce cell death. For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of the bcr-abl transcript, we applied the siRNA approach. Stable degradation of bcr-abl mRNA was achieved by using a retroviral vector with enhanced green fluorescent protein (EGFP) reporter. Despite a high (>90%) transduction efficiency we detected only a transient decrease in Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl signaling was sufficient to induce hemoglobin expression without significant cell death. These results suggest that by transiently reducing Bcr-Abl function it is possible to overcome the differentiation blockade without evoking apoptosis in CML cells and that reduced ERK activity may have a crucial role in this process.

Unregulated activation of STAT-5, ERK1/2 and c-Fos may contribute to the phenotypic transformation from myelodysplastic syndrome to acute leukaemia.

Myelodysplastic syndrome (MDS) is characterised by ineffective erythropoiesis and poor progenitor response to erythropoietin (Epo). The aim of this study was to determine the role of the Epo-R mediated signalling in the rise of MDS and whether alteration of signalling pathways contribute to the leukeamogenesis from MDS to acute leukaemia. We analysed Epo and GM-CSF induced ERK1/2 activation, c-Fos expression, STAT-5 and AP-1 DNA binding activities in mononuclear cells of umbilical cord blood (UCBMNC), normal marrow (NBMMNC) or marrow with MDS, AML with prior MDS and de novo AML. In UCBMNC and NBMMNC, Epo and GM-CSF induced the activation of STAT-5 DNA binding and ERK 1/2 activation (n = 6). In contrast, in MDS RA, both signalling pathways were activated only by GM-CSF but not by Epo (n = 7). In acute leukaemia, elevated basal activity of STAT-5 DNA binding appeared in 8/8 cases, which was independent of Epo or GM-CSF treatment. In normal and MDS samples, c-Fos and Egr-1 proteins were not detectable and the expression levels were not increased by Epo or GM-CSF treatment. In contrast, we found an elevated level of c-Fos expression in 5/8 acute leukemia cases, which was not further increased in the presence of Epo or GM-CSF. The elevated c-Fos expression was accompanied by an extremely high blast number in 5/5 cases. These results suggest that impaired ERK/MAPK activation, similarly to impaired STAT-5 activation in Epo-R signalling, may be responsible for the apoptotic process and the block of maturation in MDS RA. The results also suggest that the appearance of the constitutively activated STAT-5 DNA binding and c-Fos expression may be used as a predictor of the blastic transformation.

Activation of Raf/ERK1/2 MAP kinase pathway is involved in GM-CSF-induced proliferation and survival but not in erythropoietin-induced differentiation of TF-1 cells.

The involvement of MAPK pathways in differentiation, proliferation and survival was investigated by comparing Epo and GM-CSF signalling in human factor-dependent myeloerythroid TF-1 cells with abnormal Epo-R. GM-CSF withdrawal induced cell-cycle arrest and apoptosis accompanied by increased caspase-3 activity, DNA degradation and reduced expression of the antiapoptotic Bcl-2 and Bcl-xl proteins. Readministration of GM-CSF but not Epo reversed these processes and induced proliferation. The GM-CSF promoted cell survival and proliferation correlated with MEK-1 dependent ERK1/2, Elk-1 and CREB phosphorylation and Egr-1, c-Fos expression as well as with increased STAT-5, AP-1, c-Myb and NF-kappaB DNA-binding. In contrast, Epo failed to activate the Raf-1/ERK1/2 MAPK pathway or to induce Egr-1 and/or c-Fos expression, while it induced erythroid differentiation in GM-CSF-deprived cells. In addition, the Epo-induced haemoglobin production was inhibited in the presence of GM-CSF. These results demonstrate that the activation of MAPK cascade is not necessary for Epo-induced haemoglobin production in TF-1 cells and suggest a negative cross-talk between the signalling of GM-CSF-stimulated cell proliferation and Epo-induced erythroid differentiation.

Signalling mechanisms and the role of calcineurin in erythropoiesis.

Erythropoietin (Epo) is the principal regulator of the production of circulating erythrocytes by controlling the proliferation, the differentiation and the survival of the erythroid progenitor cells. Early down-regulation of c-myb expression in erythroleukemia cells is a common feature of the action of Epo and chemical inducers of differentiation such as DMSO. Previously we have shown that in our Epo-responsive murine erythroleukemia cell line ELM-I-1, [Ca2+]i increasing agents can mimic the effect of Epo on c-myb expression and activate nuclear signal transduction processes involved in the induction of hemoglobin synthesis. These results also indicated that the Ca2+-induced down-regulation of c-myb expression and hemoglobin synthesis are mediated by the Ca2+/calmodulin dependent serine/threonine-specific protein phosphatase PP2B, calcineurin, but the Epo induced processes are not mediated by PP2B. In spite of this, we demonstrated in this paper that in ELM-I-1 cells the Epo-induced down-regulation of c-myb expression and hemoglobin production can be effectively enhanced by the simultaneously added [Ca2+]i-increasing agent, cyclopiazonic acid (CPA). This observation further supports the existence of at least two independent signalling pathways in the mechanism of Epo and [Ca2+]i increasing agents and the strong correlation between c-myb expression and hemoglobin production in differentiating cells. Although the c-AMP-response element binding protein (CREB) could be the common target of both calcium-dependent and -independent dephosphorylation, our results do not support the involvement of CREB in the regulation of c-myb gene expression. In addition to the calcineurin mediated down-regulation of c-myb expression, we have found a negative regulatory effect in the Ca2+-mediated transcriptional activation of certain genes. In response to [Ca2+]i-increasing agents in ELM-I-1 cells, both, egr-1 and c-fos mRNA expression increased significantly after the inhibition of calcineurin by cyclosporine A. Cyclosporin A exerted stimulatory effects on the egr-1 and c-fos expression also at lower (150-400 nM) intracellular Ca2+ levels. This potential co-regulation of c-myb, egr-1 and c-fos expression by calcineurin suggests that the negative modulation of egr-1 and c-fos expression may also be important for the induction of erythroid differentiation by [Ca2+]i-increasing agents. This negative modulation may also contribute to the Epo-induced differentiation in the case of a moderate increase of [Ca2+]i.

Stimulation of the Ca2+-mediated egr-1 and c-fos expression in murine erythroleukaemia cells by cyclosporin A.

The Ca2+-induced expression of the primary response genes egr-1 and c-fos was investigated in the murine erythroleukaemia cell line ELM-I-1. Exposure of the cells to the Ca2+-ionophore A23187 led to a rapid transient rise in egr-1 and c-fos mRNA production followed by an increase in Egr-1 and c-Fos protein levels as well as an increase in Egr-1 and activator protein 1 (AP-1) DNA-binding activity. Preincubation of the cells with KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinases, strongly decreased the Ca2+-mediated expression of egr-1 and c-fos. In contrast, treatment with cyclosporin A, which inhibits the Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin, increased both egr-1 and c-fos mRNA production and the DNA-binding activity of the Egr-1 and AP-1 transcription factors in response to the intracellular Ca+ concentration ([Ca2+]i)-increasing agents A23187 or cyclopiazonic acid. Enhancement of the Ca2+-induced c-fos and egr-1 expression by cyclosporin A was correlated with the capability of this agent to inhibit calcineurin phosphatase activity in ELM-I-1 cells. Studies on the phosphorylation state and DNA-binding activity of the cAMP response element-binding protein (CREB) did not demonstrate an early Ca2+-dependent activation of this transcription factor, suggesting that the regulation of c-fos and egr-1 expression by Ca2+ is not linked to CREB in the haematopoietic ELM-I-1 cells. The results indicate that calcineurin exerts negative regulatory effects on both egr-1 and c-fos expression in murine erythroleukaemia cells, in addition to the calcineurin-mediated down-regulation of c-myb expression observed previously in this cell system. This study therefore emphasizes the important role of calcineurin as a negative modulator of gene expression in certain cell types.

Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency.

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

Signalling mechanisms in erythropoiesis: the enigmatic role of calcium.

The glycoprotein hormone, erythropoietin is the principal regulator of the production of circulating erythrocytes by controlling proliferation, differentiation and survival of its target erythroid progenitor cells. The receptor for erythropoietin is a type I cytokine receptor lacking intrinsic tyrosine kinase activity. It mediates tyrosine phosphorylation through its association with nonreceptor tyrosine kinases such as JAK2 and initiates a cascade of signalling events in response to erythropoietin. Significant progress has been made in identifying signalling pathways triggered by erythropoietin. However, the exact signalling mechanisms mediating the known physiological effects of erythropoietin in erythroid progenitor cells are poorly understood. There are many open questions including the role of Ca2+ in erythropoietin induced signal transduction. Although the results concerning the effect of erythropoietin on [Ca2+]i in various erythroid cells are conflicting, [Ca2+]i-increasing agents mimic the effect of erythropoietin on c-myb expression and activate the program of haemoglobin synthesis in murine erythroleukemia cells. An attempt is made in this review to survey recent data on the erythropoietin-induced signal transduction with respect to the different physiological effects of this hormone.

Ca2+/calmodulin-dependent and -independent down-regulation of c-myb mRNA levels in erythropoietin-responsive murine erythroleukemia cells. The role of calcineurin.

Down-regulation of c-myb mRNA levels by [Ca2+]i-increasing agents (A23187, thapsigargin, cyclopiazonic acid) and erythropoietin was comparatively studied in the erythropoietin-responsive murine erythroleukemia cell line, ELM-I-1. The Ca2+-induced suppression of c-myb mRNA could be inhibited by the calmodulin antagonists trifluoperazine and calmidazolium, as well as by cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinases, did not antagonize the Ca2+-mediated decrease in c-myb mRNA. In cyclosporin A-treated ELM-I-1 cells, a close correlation could be demonstrated between the antagonization of the Ca2+ effect on c-myb mRNA levels and inhibition of the calcineurin phophatase activity. On the other hand, FK506, which did not inhibit calcineurin activity in ELM-I-1 cells, failed to prevent the Ca2+-mediated decrease in c-myb mRNA. The erythropoietin-induced down-regulation of c-myb mRNA levels could be demonstrated also in the presence of EGTA and was resistant to calmodulin antagonists and cyclosporin A. In addition, no increase in [Ca2+]i was observed in ELM-I-1 cells in response to erythropoietin. Cyclosporin A inhibited the Ca2+-induced hemoglobin production, while the erythropoietin-mediated increase in hemoglobin synthesis was not affected. The results indicate that the Ca2+-induced decrease in c-myb mRNA and increase in hemoglobin synthesis is mediated by calcineurin, while these effects of erythropoietin occur independently of Ca2+ in ELM-I-1 cells. Calcineurin may be involved in the regulation of c-myb expression in erythroid precursor cells and Ca2+ signals via calcineurin may positively modulate the differentiation inducing action of erythropoietin.

Erythrocyte lipids in triose-phosphate isomerase deficiency.

Marked hypoalphalipoproteinemia was found together with relatively low serum cholesterol, triacylglycerol, and LDL levels in a triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1)-deficient Hungarian family, especially in the two compound-heterozygote brothers. Apart from a slight increase in palmitic and stearic acids together with a slight decrease in oleic and linoleic acids, no other changes were found in the fatty acid composition of the erythrocyte phospholipids. Anisotropy measurements with n-(9-anthroyloxy) stearic and -palmitic acid fluorophores revealed increased motional freedom of the fatty acid chains in the external lipid layers of the intact erythrocytes from all members of the TPI-deficient family as compared with normal age-matched controls. This asymmetric increase in membrane fluidity was found to be significantly higher in the propositus than in his compound-heterozygote brother without any neurological disorders. The change in membrane fluidity may result from as-yet-unresolved aspects of the lipid composition of the plasma membrane. Our findings that the differences between the TPI-deficient individuals and normal controls and the differences between the two compound-heterozygote brothers were all absent in the phospholipid extracts of the same erythrocytes favor the assumption that the increased motional freedom of the fatty acid chains in the external surface of the bilayer is caused by the binding of the mutant TPI molecule to the N-terminal sequence of band 3 protein.

Early transient suppression of c-myb mRNA levels and induction of differentiation in Friend erythroleukemia cells by the [Ca2+]i-increasing agents cyclopiazonic acid and thapsigargin.

Cyclopiazonic acid and thapsigargin, inhibitors of the endoplasmic reticulum Ca2+ pump were shown to elevate [Ca2+]i in Friend erythroleukemia cells, line F4-6, at concentrations of 1-5 microM and 0.5-2 nM, respectively. At the same concentrations, these agents induced a strong suppression of c-myb mRNA levels within 3 h, whereas c-myc expression remained unaffected. The c-myb expression recovered and approached pretreatment levels at 9-12 h of incubation. The decrease in c-myb mRNA was prevented in Ca(2+)-free medium. Treatment of F4-6 cells with EGTA led to a transient increase in c-myb mRNA with the same kinetics as the Ca2+ pump inhibitor-induced suppression, indicating that c-myb expression is bidirectionally regulated by changes in [Ca2+]i. Studies on the differentiation status of F4-6 cells following cyclopiazonic acid or thapsigargin exposure demonstrated a marked increase in beta-globin mRNA synthesis at 60h and in hemoglobin production at 96 h. These results provide further evidence that a rise in the cytosolic Ca2+ concentration is capable, in Friend erythroleukemia cells, of inducing an early transient suppression of c-myb mRNA levels, which is followed by terminal erythroid differentiation.

Ca2+ or Mg2+ nucleotide phosphohydrolases in myometrium: two ecto-enzymes.

A high level of Ca2+ or Mg2+ nucleotide phosphohydrolase activity is present on the outside surface of intact myometrial cells and is also observed in the isolated plasma membranes. About half of this activity is labile while the remainder is stable. The characteristics of the activities suggest the presence of at least two different ecto-enzymes. The stable component (Km for Ca2+ about 0.1 mM) accepts XTP or XDP as substrate, is not inhibited by p-chloromercuriphenylsulfonate or inorganic phosphate, but is inhibited by 20 mM NaN3. The labile component (Km for Ca2+ nearly 1 mM) cleaves XTP but not XDP, and is inhibited by p-chloromercuriphenyl-sulfonate and inorganic phosphate, but not by NaN3. The activity of the labile component can be restored by removing the cells from the incubation medium and resuspending them in fresh medium. This suggests that the 'lability' is due to product inhibition, probably by inorganic orthophosphate. While the Ca2+ pump of myometrial plasma membranes was inhibited by 0.1 microM oxytocin, these ecto-enzymes were unaffected by oxytocin concentrations up to 10 microM. Because of its high activity and rapid inactivation by product inhibition, the labile enzyme may be involved in the regulation of purinergic receptors.

Oxytocin pretreatment of pregnant rat uterus inhibits Ca2+ uptake in plasma membrane and sarcoplasmic reticulum.

Myometrium from rats in varying stages of pregnancy (from 17 to 22 days) was treated with oxytocin (0.1-10 microM) and plasma membranes and sarcoplasmic reticulum were isolated using a Percoll gradient. When the myometrium had been treated with oxytocin, Ca2+ uptake was reduced by 29.4% in the plasma membrane and by 32.6% in the sarcoplasmic reticulum. The inhibitory action of oxytocin was highly dependent upon the stage of gestation: Only membranes from rats close to term (21-22 days) exhibited reduced Ca2+ transport activity after hormone treatment. This effect correlated highly with a significant decrease in the serum progesterone level of these animals. In plasma membrane vesicles, oxytocin reduced the maximal velocity of the Ca2+ pump without significantly affecting the affinity for Ca2+. Oxytocin did not affect the passive permeability of the plasma membranes, nor their proportion of sealed inside-out vesicles nor the amount of Ca2(+)-pump protein in these membranes. In addition, oxytocin caused no change in the passive permeability of the membrane nor in the rate of inositol trisphosphate-induced Ca2+ release from the sarcoplasmic reticulum. These results suggest that there is a specific action of oxytocin on the activity of the myometrial plasma membrane and sarcoplasmic reticulum Ca2+ pumps which may contribute to the maintenance of an elevated intracellular calcium level during parturition.

Structurally distinct mammalian calmodulins.

Rabbits immunized with dinitrophenylated calmodulin produced monospecific antibody against CaM. Using the purified antibody, enzyme-linked immunosorbent assays (ELISAs) were carried out for calmodulin (CaM). The immunological heterogeneity of human and bovine CaMs from erythrocytes was investigated by means of indirect and inhibition ELISAs. The cross-reactivity between the two CaMs was found to be 50% in the indirect ELISA. An eight times higher concentration of human CaM was necessary to produce 30% inhibition in an inhibition assay. The effect of anti-bovine CaM on the stimulation of the red cell membrane calcium pump by human and bovine CaM has also been studied. We have found that (a) the human and bovine CaMs showed indistinguishable activator activities; (b) the antibody partially inhibited the stimulating effects of CaMs; (c) the inhibition was much less effective in the case of stimulation by human CaM. These results suggest that there is a difference in the reactivity of the anti-bovine CaM antibody with bovine and human CaMs. This difference can be attributed to a slight deviation in the antibody binding structure of the two mammalian CaMs in or near the antigenic site of the CaMs.

Thrombin-induced activation of calcium transport pathways and their role in platelet functions.

In human platelets thrombin-induced calcium release from intracellular stores, the consequent influx of extracellular calcium, as well as their role in the aggregation and ATP-secretion reactions were examined. In indo-1-loaded platelets intracellular calcium release was studied in the presence of excess EGTA in the incubation medium, while calcium influx was followed after a rapid repletion of external calcium. After thrombin-stimulation both calcium release and calcium influx produced about the same peak levels of cytoplasmic free calcium but in the first case it was only a transient response, while in the latter one a sustained calcium signal was observed. Increased calcium influx could be evoked for several minutes after the addition of thrombin, it was selectively inhibited by Mg2+ (20 mM) and Ni2+ (1 mM) ions, by neomycin and by PCMB, a non-penetrating SH-group reagent. This calcium influx was practically insensitive to organic calcium channel blockers. Thrombin-induced platelet aggregation was only partial in the absence of external calcium, even if excess magnesium was present in the media, while the aggregation response became complete if external calcium was repleted. A significantly reduced aggregation could be seen in calcium-containing media if calcium influx was selectively inhibited. Platelet ATP-secretion under the same conditions did not depend on external calcium or on calcium influx. These data indicate that in thrombin-stimulated platelets the opening of specific plasma membrane calcium channels can be selectively modulated and these channels play a major role in the development of a full-scale aggregation.

Effects of phosphoinositides on calcium movements in human platelet membrane vesicles.

In a mixed endoplasmic and surface-type membrane vesicle preparation from human platelets the polyphosphoinositides PIP and PIP2, similarly to IP3, were found to induce a rapid calcium release reaction. At physiological (resting) cytoplasmic calcium concentrations (0.1-0.3 microM) the PIP2 and IP3 concentrations producing half-maximum calcium release were similar (0.7 microM) and both agents could mobilize about 30-40% of the intravesicular calcium. However, the phosphodiesteric degradation of PIP2 in the membrane vesicles was found to be negligible and the ion- and drug-sensitivities of the calcium release reactions were different. The IP3-induced calcium release was selectively inhibited by micromolar calcium concentrations and by cinnarizine, while the PIP2-induced release was blocked by magnesium ions and neomycin. The calcium release evoked by either agent was inhibited by low concentrations of lanthanum but, in contrast to the ATP-dependent calcium pump, it was insensitive to vanadate, quercetin and to the lowering of the incubation temperature. When added simultaneously or in a rapid succession, maximum effective IP3 and PIP2 concentrations produced an additive calcium release reaction. Based on these data we suggest that IP3 and PIP2, respectively, induce rapid transmembrane calcium movements involving different transport pathways and/or membrane calcium pools, which are not related to the active calcium transport systems.