BCR/ABL preleukemic fusion gene in subpopulations of hematopoietic stem and progenitor cells from human UCB.

The BCR/ABL preleukemic fusion gene (PFG) is one of the most frequent fusion genes in acute lymphoblastic leukemia (ALL) and was also detected in hematopoietic cells from umbilical cord blood (UCB) of healthy newborns. Since hematopoietic stem/progenitor cells (HSPC) are considered to be a critical cellular target for origination of leukemia, we have studied the presence of BCR/ABL PFG in expanded subpopulations of HSPC and differentiated cells from UCB of those healthy newborns, who have previously been tested positive for BCR/ABL by screening of their UCB mononuclear cells using RT-qPCR and FISH methods. We isolated cells from human UCB samples positive for BCR/ABL and negative controls. The isolated cells were sorted into 5 hematopoietic and progenitor cell subpopulations. We analyzed BCR/ABL in sorted and expanded subpopulations of UCB using FISH and RT-qPCR. We found that the number of BCR/ABL positive cells was similar in each studied subpopulation and the same as in differentiated lymphocytes. Our data showed that there is no specific subpopulation of hematopoietic and progenitor stem cells with an increased leukemogenic potential due to the presence of higher copies of BCR/ABL.

Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.

In Arabidopsis the plasma membrane nitrate transceptor (transporter/receptor) NRT1.1 governs many physiological and developmental responses to nitrate. Alongside facilitating nitrate uptake, NRT1.1 regulates the expression levels of many nitrate assimilation pathway genes, modulates root system architecture, relieves seed dormancy and protects plants from ammonium toxicity. Here, we assess the functional and phenotypic consequences of point mutations in two key residues of NRT1.1 (P492 and T101). We show that the point mutations differentially affect several of the NRT1.1-dependent responses to nitrate, namely the repression of lateral root development at low nitrate concentrations, and the short-term upregulation of the nitrate-uptake gene NRT2.1, and its longer-term downregulation, at high nitrate concentrations. We also show that these mutations have differential effects on genome-wide gene expression. Our findings indicate that NRT1.1 activates four separate signalling mechanisms, which have independent structural bases in the protein. In particular, we present evidence to suggest that the phosphorylated and non-phosphorylated forms of NRT1.1 at T101 have distinct signalling functions, and that the nitrate-dependent regulation of root development depends on the phosphorylated form. Our findings add to the evidence that NRT1.1 is able to trigger independent signalling pathways in Arabidopsis in response to different environmental conditions.

Constitutive 53BP1/γH2AX foci are increased in cells of ALL patients dependent on BCR-ABL and TEL-AML1 preleukemic gene fusions.

Childhood leukemia arises from hematopoietic stem cells by induction of mutations. Quite often chromosomal translocations arise prenatally as first key event in multistage process of leukemogenesis. These translocations result in so called preleukemic gene fusions (PGFs), such as BCR-ABL and TEL-AML1, which generate hybrid proteins with altered properties. Critical DNA damage resulting in translocations are DNA double-strand breaks (DSBs). BCR-ABL and TEL-AML1 were shown to be associated with increased constitutive DSBs in various model systems. We analyzed cells from peripheral blood and CD34-/CD34+ cells from bone marrow of pediatric acute lymphoblastic leukemia (ALL) patients harboring BCR-ABL or TEL-AML1. We used sensitive technique that is based on automated enumeration of DSB co-localizing proteins γH2AX and 53BP1, which form so called DNA repair foci. We found that level of constitutive γH2AX/53BP1 foci is significantly higher in cells of ALL pediatric patients than in healthy subjects. There was also significant increased level of constitutive γH2AX/53BP1 foci in cells from ALL patients harboring BCR-ABL or TEL-AML1 compared to patients without PGFs. The same increase was observed regardless cell type for both PGFs. Our data on increased DSB levels in the BCR-ABL/TEL-AML1 patient's cells support a model where BCR-ABL/TEL-AML1 induces DNA instability through facilitating mutagenesis and appearance of additional genetic alterations driving leukemogenesis.

TNF signaling: early events and phosphorylation.

Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.

Induction of tumor necrosis factor alpha in murine macrophages with various strains of Coxiella burnetii and their lipopolysaccharides.

The ability of various strains of Coxiella burnetii (C.b.) and their phase I and II lipopolysaccharides (LPSs) to induce tumor necrosis factor alpha (TNF-alpha) in peritoneal Balb/c mouse macrophages in vitro was investigated. Considerable differences in the induction ability were observed in dependence on the strain applied. In a TNF-alpha bioassay, the most effective inducers were both corpuscles and LPSs of the strains Priscilla and Scurry, followed by Nine Mile, Luga, and Henzerling I. In contrast, in ELISA, the most effective inducers were LPSs of the strains Luga and Henzerling, followed by Nine Mile, Priscilla, and Scurry. The role of toll-like receptor 4 (TLR4) in the induction was confirmed by the use of C3H/HeJ mouse macrophages. Thus, the induction of TNF-alpha was much higher in Balb/c mouse macrophages than that in TLR4-deficient C3H/HeJ mouse macrophages. Differences in the results of the bioassay and those of ELISA suggest a role of another secreted factor(s) induced with C.b. in murine macrophages that could act synergically with TNF-alpha in L929 cells in the bioassay. The observed differences in TNF-alpha induction might play a role in the pathobiology of Q fever.

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity.

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.

Knock-out of the neural death effector domain protein PEA-15 demonstrates that its expression protects astrocytes from TNFalpha-induced apoptosis.

Apoptosis is a very general phenomenon, but only a few reports concern astrocytes. Indeed, astrocytes express receptors for tumor necrosis factor (TNF) alpha, a cytokine demonstrated on many cells and tissues to mediate apoptosis after recruitment of adaptor proteins containing a death effector domain (DED). PEA-15 is a DED-containing protein prominently expressed in the CNS and particularly abundant in astrocytes. This led us to investigate if PEA-15 expression could be involved in astrocytic protection against deleterious effects of TNF. In vitro assays evidence that PEA-15 may bind to DED-containing protein FADD and caspase-8 known to be apical adaptors of the TNF apoptotic signaling. After generation of PEA-15 null mutant mice, our results demonstrate that PEA-15 expression increases astrocyte survival after exposure to TNF.

Endothelin induces a calcium-dependent phosphorylation of PEA-15 in intact astrocytes: identification of Ser104 and Ser116 phosphorylated, respectively, by protein kinase C and calcium/calmodulin kinase II in vitro.

PEA-15 (phosphoprotein enriched in astrocytes, Mr = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 microM) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser104, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser116, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser116 and had no effect on Ser104, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.

Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse.

Axonal navigation during development requires that cues present in the extracellular environment be capable of modifying the structure of the cone in a dynamic way. Protein kinase C (PKC) has long been suspected to be one of the multiple molecular relays present in the terminal structure of the developing axon and involved in the transduction of extracellular signals. The latter proposal is, however, based on the use of drugs or of protocols leading to pleiotropic and often nonspecific effects. In the present study, we have taken advantage of the discovery of a peptide capable of translocating across biological membranes and to accumulate in the cytoplasm and nucleus of cells in culture, to internalize a highly specific peptidic inhibitor of PKC. We demonstrate that linking the two peptides (vector and PKC inhibitor) allows the internalization of the latter in live cells, specifically inhibits PKC and provokes a rapid modification of growth cone morphology. This set of data thus establishes that a peptidic inhibitor of PKC activity, once internalized, provokes a change in growth cone morphology, reminiscent of the collapse phenotype. In addition, the present study describes a new efficient and harmless way to introduce pharmacologically active substances in neural cells in culture.

Salivary gland extracts of partially fed Dermacentor reticulatus ticks decrease natural killer cell activity in vitro.

The salivary glands and saliva of ticks (Arachnida, Acari, Ixodida) play a vital role in blood feeding, including manipulation of the host's immune response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva. A factor synthesized in the salivary glands of feeding ticks potentiates the transmission of certain tick-borne viruses. We show that salivary gland extracts (SGE) derived from Dermacentor reticulatus female ticks fed for 6 days on laboratory mice (SGED6) induced a decrease in the natural killer (NK) activity of effector cells obtained from 16 healthy blood donors. The decreased activity ranged from 14 to 69% of NK activity observed with the respective untreated effector cells. Such a decrease was not observed after treatment of effector cells with SGE from unfed ticks. Ten-fold dilution of SGED6 significantly reduced the capacity to decrease NK activity and a further 10-fold dilution almost eliminated the effect. After addition of IFN-alpha 2, the SGED6-induced decrease in NK activity was restored to activity levels approaching those of untreated cells. The apparent reversibility of the inhibition indicates that the effect of SGED6 on NK activity was not due to cytotoxicity. The results demonstrate the presence of a factor(s) in the salivary gland products of feeding D. reticulatus female ticks that influences human NK activity in vitro. These data suggest a possible mechanism by which tick SGE potentiates the transmission of some tick-borne viruses through suppression of NK activity.

Cross-species antiviral and antiproliferative activity of human interferon-omega.

The antiviral and antiproliferative activities of human interferon-omega (IFN-omega) on two human cell lines and on VERO (monkey), MDBK (calf), SPEV (pig), L929 (mouse), BHK-21 (hamster), and MDCK (dog) cell lines were compared with those of human IFN-alpha 1 and IFN-alpha 2. The results are tabulated. Compared with its antiviral titer on human A549 cells, INF-omega was more active on mouse cells and even more active on the pig cells, but had little activity on the hamster cells and virtually none on the dog cells. IFN-omega also inhibited the growth of all these cells to a greater or lesser extent, and there was in general an apparent correlation between its antiviral and antiproliferative activities on the different cells, except that the dog cells were relatively much more sensitive to the antiproliferative effect.

[Interferons as antineoplastic drugs--direct regulatory effects on tumor cells]. / Interferóny ako protinádorové liecivo--priamy regulacný efekt na nádorové bunky.

Interferons (IFNs) are a family of pleiotropic cell regulatory molecules and members of the cytokine network. Apart from a central role in the body's first-line defence against infections, they are important in regulation of immune responses and may be involved in control of cell growth and differentiation. Over the past ten years, clinical trials provided unequivocal evidence that IFNs to have anticancer activity, even though this is valid in a restricted range of tumours mainly of haematopoietic origin. We currently suppose three ways in which IFNs could affect tumour growth: 1. Direct regulatory effects on tumour cells. 2. Immunomodulatory effects that enhance or even initiate a host response to the tumour. 3. Regulatory effects on host/tumour interaction not associated with immune responses. We reviewed the direct regulatory effects of IFNs on tumour cells and results of clinical trials of IFNs in cancer therapy. (Tab. 2, Ref. 85.)

Masking of HLA class I molecules expressed on K-562 target cells can restore their susceptibility to NK cell cytolysis.

The aim of our study was to follow whether class I HLA antigens play a role in the susceptibility of K-562 target cells to NK cell lysis as well as in the interaction between NK and target cells. After K-562 cells were treated with rIFN-gamma, HLA class I antigens appeared on their surface. Those HLA class I+, in comparison to HLA class I-, K562 target cells became about 40-50% less susceptible to NK cell cytolysis. This influence of HLA class I antigens was abrogated when class I+ K-562 cells were incubated with anti-HLA class I monoclonal antibodies Bra 23/9. The restoration of susceptibility of these targets to NK cell cytolysis was not caused by ADCC mechanism as determined by F(ab')2 fragments or by mAb CD16. Further, the percentage of conjugates between effector and target cells was not significantly altered neither when HLA class I antigens appeared on the surface of K-562 target cells nor when HLA class I+ K-562 cells were incubated with anti-HLA class I monoclonal antibodies. To study this question, we used a different approach because both expression of HLA class I molecules and their masking with appropriate monoclonal antibody were done by means of the same cell line of target cells.

In vitro antiproliferative effect of interferon alpha in solid tumors: a potential predictive test.

An in vitro test for the antiproliferative effect of human leukocyte interferon (IFN-alpha) was performed in primary cultures of tumor cells obtained from 32 patients with either malignant melanoma (13), renal carcinoma (4) or bladder carcinoma (15). Our results demonstrated activity of IFN in all three groups of solid tumors. However, appreciable differences in sensitivity to antiproliferative effect of IFN between individual tumors of the same type were found. The potential of this antiproliferative test for prediction of treatment response in IFN-therapy is discussed.

Relationship between depression of HBsAg production and DNA synthesis by interferons in human hepatoma cell line.

The influence of different interferons (IFNs) on HBsAg production and DNA synthesis was studied in PLC/PRF/5 cells using 30 I.U./ml of natural HuIFN-alpha, 25 I.U./ml of recombinant HuIFN-alpha 2, and 5 I.U./ml of natural murine IFN-alpha/beta. All three IFN types inhibited significant inhibitory effect on HBsAg production during the second 24 hr-interval following their addition. After 96 hr HBsAg production had returned to normal levels. Natural HuIFN-alpha clearly depressed cellular DNA synthesis 24 hr after IFN addition which returned to normal within the next 24 hr. Recombinant HuIFN-alpha 2 influenced DNA synthesis only slightly and the mouse IFN-alpha/beta showed no effect.