Leukemia stem cell immunophenotyping tool for diagnostic, prognosis, and therapeutics.

The existence of cancer stem cells is debatable in numerous solid tumors, yet in leukemia, there is compelling evidence of this cell population. Leukemic stem cells (LSCs) are altered cells in which accumulating genetic and/or epigenetic alterations occur, resulting in the transition between the normal, preleukemic, and leukemic status. These cells do not follow the normal differentiation program; they are arrested in a primitive state but with high proliferation potential, generating undifferentiated blast accumulation and a lack of a mature cell population. The identification of LSCs might guide stem cell biology research and provide key points of distinction between these cells and their normal counterparts. The identification and characterization of the main features of LSCs can be useful as tools for diagnosis and treatment. In this context, the aim of the present review was to connect immunophenotype data in the main types of leukemia to further guide technical improvements.

P2X(7) receptor in the kidneys of diabetic rats submitted to aerobic training or to N-acetylcysteine supplementation [corrected].

Previous studies in our laboratory showed that N-acetylcysteine supplementation or aerobic training reduced oxidative stress and the progression of diabetic nephropathy in rats. The P2X(7 receptor is up-regulated in pathological conditions, such as diabetes mellitus. This up-regulation is related to oxidative stress and induces tissue apoptosis or necrosis. The aim of the present study is to assess the role of P2X(7) receptor in the kidneys of diabetic rats submitted to aerobic training or N-acetylcysteine supplementation. Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, i.v.) and the training was done on a treadmill; N-acetylcysteine was given in the drinking water (600 mg/L). By confocal microscopy, as compared to control, the kidneys of diabetic rats showed increased P2 × 7 receptor expression and a higher activation in response to 2'(3')-O-(4-benzoylbenzoyl) adenosine5'-triphosphate (specific agonist) and adenosine triphosphate (nonspecific agonist) (all p<0.05). All these alterations were reduced in diabetic rats treated with N-acetylcysteine, exercise or both. We also observed measured proteinuria and albuminuria (early marker of diabetic nephropathy) in DM groups. Lipoperoxidation was strongly correlated with P2X(7) receptor expression, which was also correlated to NO•, thus associating this receptor to oxidative stress and kidney lesion. We suggest that P2X(7) receptor inhibition associated with the maintenance of redox homeostasis could be useful as coadjuvant treatment to delay the progression of diabetic nephropathy.

Nitric oxide-induced murine hematopoietic stem cell fate involves multiple signaling proteins, gene expression, and redox modulation.

There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO(•)), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO(•) , one produced by endothelial cells stimulated with carbachol in vitro and another using the NO(•)-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in vivo. Two main NO(•) effects were observed: proliferation of HSCs-especially of the short-term HSCs-and its commitment and terminal differentiation to the myeloid lineage. NO(•)-induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki-67, upregulation of Notch-1, Cx43, PECAM-1, CaR, ERK1/2, Akt, p38, PKC, and c-Myc. NO(•)-induced HSCs differentiation was characterized by the increase in granulocytic-macrophage progenitors, granulocyte-macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA-3 and Ikz-3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO(•) in inducing HSCs proliferation and myeloid differentiation involving multiple signaling.

PLCγ2 and PKC are important to myeloid lineage commitment triggered by M-SCF and G-CSF.

Myeloid differentiation is a complex process whereby mature granulocytes or monocytes/macrophages are derived from a common myeloid progenitor through the coordinated action of hematopoietic cytokines. In this study, we explored the role of the Ca(2+)i signaling transduction pathway in the commitment of hematopoietic stem/progenitor cells to either the monocytic or granulocytic lineage in response to macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). M-CSF and G-CSF induce cell expansion and monocyte or granulocyte differentiation, respectively, without affecting the percentage of hematopoietic progenitor cells. Colony-forming units (CFUs) and flow cytometry demonstrated the involvement of phospholipase Cγ (PLCγ) and protein kinase C (PKC) in monocyte/granulocyte commitment. In addition, using flow cytometry and RNA interference, we identified PLCγ2 as the PLCγ isoform that participates in this cell expansion and differentiation. Differences in signaling elicited by M-CSF and G-CSF were observed. The M-CSF-related effects were associated with the activation of ERK1/2 and nuclear factor of activated T-cells (NFAT); the inhibition of both molecules reduced the number of colonies in a CFU assay. In contrast, using flow cytometry and confocal evaluation, we demonstrated that G-CSF activated Jak-1 and STAT-3. Additionally, the effects induced by G-CSF were also related with the participation of Ca(2+) calmodulin kinase II and the transcription factor PU.1. STAT-3 activation and the increase of PU.1 expression were sensitive to PLC inhibition by U73122. These data show that PLCγ2 and PKC are important upstream signals that regulate myelopoiesis through cytokines, and differences in M-CSF and G-CSF downstream signaling were identified.

Cell-permeable gomesin peptide promotes cell death by intracellular Ca(2+) overload.

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 µM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.

Requirement for PLCγ2 in IL-3 and GM-CSF-stimulated MEK/ERK phosphorylation in murine and human hematopoietic stem/progenitor cells.

Even though the involvement of intracellular Ca(2+) Ca(i)(2+) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3) ) levels and Ca(i)(2+) release in murine and human hematopoietic stem/progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+) -dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells.

Angiotensin II binding to angiotensin I-converting enzyme triggers calcium signaling.

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.

Heparin induces rat aorta relaxation via integrin-dependent activation of muscarinic M3 receptors.

Previous reports have shown that heparin may promote human hypotension and vascular relaxation by elevation of NO levels through unclear mechanisms. We hypothesized that endothelial muscarinic M(3) receptor activation mediates the heparin-induced vasodilation of rat aortic rings. The experiments were carried out using unfractionated heparin extracted from bovine intestinal mucosa, which elicited an endothelium and NO-dependent relaxation of aortic segments with maximal potency and efficacy (EC(50): 100±10 µmol/L; E(max): 41±3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M(3) muscarinic receptor is involved in this phenomenon. However, no direct binding of heparin to muscarinic receptors was observed. More importantly, studies performed using the arginine-glycine-aspartic acid peptide and 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine, an Src family inhibitor, reduced by 51% and 73% the heparin-dependent relaxation, respectively, suggesting the coupling of heparin and M(3) receptor through extracellular matrix molecules and integrin. Furthermore, unfractionated heparin induced activation of focal adhesion protein kinase, Src, and paxillin. Finally, fluorescence resonance energy transfer approach confirmed the interaction of the M(3) receptor to integrin. Taken together, these data demonstrate the participation of M(3) receptor and integrin in heparin-dependent relaxation of vascular smooth muscle. These results provide new insights into the molecular mechanism and potential pharmacological action of heparin in vascular physiology.

Myelopoiesis modulation by ACE hyperfunction in kinin B(1) receptor knockout mice: relationship with AcSDKP levels.

Angiotensin I-converting enzyme (ACE), a common element of renin-angiotensin system (RAS) and kallikrein-kinin system (KKS), is involved in myelopoiesis modulation, mainly by cleaving the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Based on this finding and in our results showing B1 and B2 kinin receptors expression in murine bone marrow (BM) cells, we evaluated the ACE influence on myelopoiesis of kinin B1 receptor knockout mice (B1KO) using long-term bone marrow cultures (LTBMCs). Captopril and AcSDKP were used as controls. Enhanced ACE activity, expressed by non-hematopoietic cells (Ter-199(-) and CD45(-)), was observed in B1KO LTBMCs when compared to wild-type (WT) cells. ACE hyperfunction in B1KO cells was maintained when LTBMCs from B1KO mice were treated with captopril (1.0microM) or AcSDKP (1.0nM). Although no alterations were observed in ACE mRNA and protein levels under these culture conditions, 3.0nM of AcSDKP increased ACE mRNA levels in WT LTBMCs. No alteration in the number of GM-CFC was seen in B1KO mice compared to WT animals, even when the former were treated with AcSDKP (10microg/kg) or captopril (100mg/kg) for 4 consecutive days. Hematological data also revealed no differences between WT and B1KO mice under basal conditions. When the animals received 4 doses of lipopolysaccharide (LPS), a decreased number of blood cells was detected in B1KO mice in relation to WT. We also found a decreased percentage of Gr1(+)/Mac-1(+), Ter119(+), B220(+), CD3(+), and Lin(-)Sca1(+)c-Kit(+) (LSK) cells in the BM of B1KO mice compared to WT animals. Low AcSDKP levels were observed in BM cultures from B1KO in comparison to WT cultures. We conclude that ACE hyperfunction in B1KO mice resulted in faster hydrolysis of AcSDKP peptide, which in turn decreased in BM tissues allowing HSC to enter the S stage of the cell cycle.

Low-intensity pulsed ultrasound-dependent osteoblast proliferation occurs by via activation of the P2Y receptor: role of the P2Y1 receptor.

Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of fractures and nonunion-promoting acceleration of healing fractures. In this report, we investigated the implication of the P2 receptors in osteoblast proliferation induced with LIPUS treatment. We observed that ADP, ATP, UTP, and UDP promote osteoblast increase and an increase of intracellular Ca(2+), through activation of P2Y receptors. Osteoblasts' expression of the P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(12), and P2Y(13) receptors was confirmed. In addition, the participation of the P2Y(1) receptor in osteoblast increase and the ADP-dependent increase of Ca(2+) concentration were shown. Furthermore, release of ATP/purines was induced by LIPUS treatment. Finally, LIPUS-dependent osteoblast increase was abolished in the presence of the Ca(2+) chelator (BAPTA), the inositol 1,4,5-trisphosphate receptor antagonist (2-APB), and the selective P2Y(1) receptor antagonist (MRS2179). In conclusion, LIPUS treatment induces osteoblastogenesis via the release of purines, such as ATP, activating P2Y receptors, mainly the P2Y(1) receptor.

Pre-clinical antitumour evaluation of Biphosphinic Palladacycle Complex in human leukaemia cells.

Previous studies reported by our group have introduced a new antitumoural drug called Biphosphinic Palladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60 and Jurkat), but not in normal human lymphocytes. IC(50) values obtained for both cell lines using the MTT and trypan blue exclusion assays 5h after BPC treatment were lower than 8.0 microM. Using metachromatic fluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Furthermore, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatin condensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lysosomal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation, which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We conclude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptotic signalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no toxic effects for human lymphocytes were observed, we suggest that BPC is more selective for transformed cells, mainly due to their exacerbated lysosome expression.

Changes in intracellular Ca2+ levels induced by cytokines and P2 agonists differentially modulate proliferation or commitment with macrophage differentiation in murine hematopoietic cells.

The role of intracellular Ca2+ (Ca2+i) on hematopoiesis was investigated in long term bone marrow cultures using cytokines and agonists of P2 receptors. Cytokines interleukin 3 and granulocyte/macrophage colony stimulator factor promoted a modest increase in Ca2+i concentration ([Ca2+]i) with activation of phospholipase Cgamma, MEK1/2, and Ca2+/calmodulin kinase II. Involvement of protein kinase C was restricted to stimulation with interleukin 3. In addition, these cytokines promoted proliferation (20 times) and an increase in the Gr-1(-)Mac-1+ population with participation of gap junctions (GJ). Nevertheless ATP, ADP, and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), a reduction in the primitive Gr-1(-)Mac-1(-)c-Kit+ population, and differentiation into macrophages without participation of GJ. It is likely that Ca2+i participates as a regulator of hematopoietic signaling: moderate increases in [Ca2+]i would be related to cytokine-dependent proliferation with participation of GJ, whereas high increases in [Ca2+]i would be related to macrophage differentiation without maintenance of the primitive population.

Cell invasion by Trypanosoma cruzi amastigotes of distinct infectivities: studies on signaling pathways.

Trypanosoma cruzi metacyclic trypomastigotes of the major phylogenetic lineages use specific signaling pathways to invade host cells. Using a panel of drugs, we studied if the differences in the ability of extracellular amastigotes (EA) from G (T. cruzi I) and CL (T. cruzi II) strains to invade host cells could be associated to activation of specific signaling routes. Sonicated extracts from G or CL strain EA induced transient raises in HeLa cell intracellular Ca(2+) levels in a dose-dependent manner. Treatment of EA with drugs that affect Ca(2+) release from inositol-1,4,5-triphosphate-sensitive stores did not significantly affect the infectivity of either strain, whereas EA of both strains treated with ionomycin plus NH(4)Cl or nigericin that release Ca(2+) from acidocalcisomes had their infectivity reduced. Treatment of parasites with adenylate cyclase activator forskolin increased the infectivity of both strains towards HeLa cells. These data, taken together, suggest that, for host cell invasion, G and CL strain EA engage signaling pathways that lead to an increase of cyclic adenosine monophosphate and Ca(2+) mobilization from acidocalcisomes. Moreover, treatment of EA with genistein reduced by approximately 45% the invasion of HeLa cells by G but not by CL strain, implicating a protein tyrosine kinase in the process. In line with this, HeLa cell extracts contained a protein tyrosine kinase activity that mediated the phosphorylation of 87- and 175-kDa polypeptides of EA from G but not from CL strain. Regarding the target cell response, the activation of host PI3 kinase appears to be required for invasion by either strain as treatment of HeLa cells with wortmannin reduced EA infectivity. These data overall reinforce the concept that cell invasion by T. cruzi EA markedly differs from the process involving metacyclic trypomastigotes.

Aging-related changes of intracellular Ca2+ stores and contractile response of intestinal smooth muscle.

In this study, we investigated the effect of aging on intracellular Ca2+ stores, as sarcoendoplasmic reticulum (SR) and mitochondria, and the influence of these compartments on contraction of rat colon smooth muscle [Bitar, K.N., 2003. Aging and neural control of the GI tract V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins. Am. J. Physiol. Gastrointest. Liver. Physiol. 284(1), G1-G7; Marijic, J., Li, Q.X., Song, M., Nishimaru, K., Stefani, E., Toro, L., 2001. Decreased expression of voltage-and Ca2+-activated K+ channels in coronary smooth muscle during aging. Circ. Res. 88, 210-234; Rubio, C., Moreno, A., Briones, A. Ivorra, M.D., D'Ocon, P., Vila, E., 2002. Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40(6), 832-840]. Calcium stores and contraction were evaluated by simultaneous measurements of fluorescence and tension in smooth muscle strips loaded with fura-2. Results showed that activation of muscarinic receptors by methylcholine (MCh, 10 microM), induced a greater contraction in aged rats than in adult animals. The inhibition of Ca2+ ATPase by thapsigargin (TG, 1 microM) did not prevent the refilling of SR either in adult or aged rats. MCh, in the presence of TG, induced an increase in transient fluorescence, indicating a release of Ca2+ from TG-insensitive compartment. The mitochondrial uncoupler, FCCP (5 microM), caused a greater increase in intracellular Ca2+ and tension in aged rats, indicating that mitochondria may accumulate more Ca2+ during aging. The present results show that changes in intracellular Ca2+ stores, such as mitochondria and SR, affect contraction and may cause dysfunctions during aging that could culminate in severe alterations of Ca2+ homeostasis and cell damage.

Evidence for changes in the tachyphylactic property of recombinant angiotensin II AT(1) receptor expressed in CHO cells.

The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT(1) receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT(1A) receptor gene, as well as 56 bp of its 3'- and 52 bp of its 5'-untranslated regions. These cells (CHO-AT(1)) responded to angiotensin II by increases in intracellular Ca(2+) concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys(2)]angiotensin II), this analogue induced responses in CHO-AT(1) cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys(2)]angiotensin II after transfection with the angiotensin II AT(1) receptor gene. Our findings suggest that posttranscriptional control directed by the 3'- or the 5'-untranslated regions in the angiotensin II AT(1) receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis.