Total Tyrosine Phosphorylation Research Articles

IgE cross-linking induces activation of human and mouse mast cell progenitors

The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation invivo, we hypothesized that they are activated by IgE cross-linking. Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.

The effect of acrylamide on sperm oxidative stress, total antioxidant levels, tyrosine phosphorylation, and carboxymethyl-lysine expression: A laboratory study.

BackgroundAcrylamide (AA) is a reactive molecule produced during food processing at temperatures above 120∘C.ObjectiveTo evaluate the impact of different concentrations of AA on human sperm parameters, oxidative stress and total antioxidant capacity (TAC).Materials and Methods In this laboratory study, semen samples were obtained from healthy donors referred to the Taleghani Hospital, Tehran, Iran between June and July 2019. Samples were divided into four groups (n = 10/each): one control and three treatment groups (0.5, 1, and 2 mM of AA). After 2 hr of exposure to AA, the superoxide dismutase and malondialdehyde levels were measured based on colorimetric methods. The TAC was determined by the ferric-reducing antioxidant power assay. Flow cytometry was performed to measure the intracellular reactive oxygen species generation. Also, immunohistochemistry was done to determine the effect of AA on tyrosine phosphorylation and carboxymethyl-lysine expression.ResultsResults of the study demonstrated that the motility and viability of spermatozoa were significantly decreased after AA exposure (p 0.001). This decrease was also seen in the TAC and superoxide dismutase activity as well as in the phosphotyrosine percentage compared with the control (p 0.01). However, the carboxymethyl-lysine and prooxidant activity including reactive oxygen species generation and lipid peroxidation level increased (p 0.001).Conclusion Overall, the results confirmed the detrimental effect of AA on human spermatozoa which may be due to oxidative stress and decreased total antioxidant levels. AA may reduce fertility by reducing sperm capacitation and motility.

Integrin alpha6-Deletion Induces Apoptosis and Src Signaling Changes in Acute Lymphoblastic Leukemia Cells

After induction chemotherapy, the detection of minimal residual disease (MRD) in the bone marrow of patients with acute lymphoblastic leukemia (ALL) is a negative prognostic factor. Coustan-Smith et al recently identified integrin alpha6 (Itga6) as a novel MRD marker which was included in a panel of 22 flow cytometry markers for MRD ALL cells. Our data supports a role of Itga6 as a critical molecule in drug resistance of ALL. In a xenograft model of primary pre-B-ALL, a novel non-humanized antibody targeting Itga6, P5G10, significantly prolonged survival of leukemic mice after combination with chemotherapy (Vincristine, Dexamethasone, L-Asparaginase) (n=6) compared to chemotherapy only treated mice (n=6) (MST= 185 days vs. MST=71 days; p=0.0012). In vitro treatment of primary pre-B-ALL cells adhering to laminin-1 with the anti-human Itga6 P5G10 antibody caused loss of viability, suggesting that Itga6 signaling has a pro-survival function for these cells. To investigate this further, we established murine ERT2-Cre x Itga6fl/fl BCR-ABL1+ pre-B ALL cells in which Itga6 function can be ablated by tamoxifen (TAM) treatment in vitro and in vivo after injection in NSG mice. In vivo ablation of Itga6 prolonged survival of recipient leukemic mice compared to Itga6 competent leukemia recipient mice. Combination of tyrosine kinase inhibition with Itga6 ablation in vivo prolonged survival significantly compared to tyrosine kinase only treated mice (MST=127 days (n=6) vs. MST of 39.5 days (n=5); p=0.0018). When ERT2-Cre x Itga6fl/fl B-ALL cells were treated with TAM in vitro, Itga6 expression was lost over at least 5 days. Interestingly, this corresponded with a progressive decrease in cell viability. As Src-family kinases and other tyrosine kinases play important roles in signaling of integrins, we investigated specific changes in total tyrosine phosphorylation in Itga6 deleting cells to understand the underlying mechanism for Itga6 deletion associated apoptosis. Compared to empty vector x Itga6fl/fl pre-B ALL cells, cells losing Itga6 had increased signals on pTyr-containing proteins within 24 hours. To identify the affected proteins, we performed proteomic pTyr analysis and identified increased tyrosine phosphorylation on the docking protein CASL, which assembles signal transduction complexes including integrins, and decreases in phosphorylation of specific Y residues for Src family kinase substrates. Interestingly, increases in pY508 and pY531 in the non-receptor hematopoietic tyrosine kinases Lyn and Fyn compared to non-Itga6-deleted ALL cells were also noted. Lyn and Fyn are tyrosine residues and their phosphorylation inhibits the activity of these tyrosine kinases. Thus, kinases with increased phosphorylation on these residues have reduced tyrosine kinase activity. Treatment of Itga6fl/fl B-ALL cells with nilotinib, to inhibit the tyrosine kinase activity of BCR/ABL1 expressed in these cells, also caused a marked increase in pY531 Fyn levels. To further address the significance of Fyn and Lyn, we treated WT and Itga6-ablated B-ALL cells with siRNA against Lyn and Fyn. Knockdown of Lyn significantly decreased viability of Itga6 WT B-ALL cells, and combined loss of Itga6 and Lyn further lowered viability. Taken together, these results implicate Lyn and Fyn with decreased viability that accompanies Itga6 deletion. Identification of Lyn and Fyn critical for transducing Itga6 signals in B-ALL will not only yield new information on the specific function Itga6, which may be useful to others who study malignancies that involve Itga6, but will also provide a rationale on which to base possible combination treatments with Itga6 antibodies. DisclosuresNo relevant conflicts of interest to declare.

Hydrogen Peroxide Has Adverse Effects on Human Sperm Quality Parameters, Induces Apoptosis, and Reduces Survival.

Background:One of the causes of male fertility disorders is the exposure of oxidative stress on the human sperm. Understanding the mechanism of disturbance is important to develop a better treatment for infertile or subfertile patients.Aims:The aim of this study was to analyze the effects of hydrogen peroxide (H2O2) on human sperm quality parameters and cell survival.Settings and Design:This study used an experimental design.Materials and Methods:Sperm cells from 15 donors were washed in a Percoll gradient and dissolved in Biggers, Whitter, and Whittingham medium. Cells were incubated with H2O2 at various concentrations from 0 to 250 μM for 2 h. Sperm viability was examined by eosin assay, sperm kinetic by computer-assisted sperm analyzer, sperm penetration by cervical mucus penetration assay, and membrane integrity by hypo-osmotic swelling test. Sperm capacitation, apoptosis, and cell survival were analyzed using western immunoblotting.Statistical Analysis Used:One-way ANOVA on SPSS 21 combined with post hoc LSD test was used to analyze differences among the groups. A P < 0.05 was considered significant.Results:Sperm viability and kinetic were significantly reduced at H2O2 concentrations of 200 and 250 μM. H2O2 reduced sperm capability to penetrate cervical mucus and also damage cell membrane integrity at all concentrations used. H2O2 significantly inhibited sperm capacitation, indicated by reduced total tyrosine phosphorylation. H2O2 exposure stimulated activation of caspase 3 and significantly reduced phosphorylated AKT at all concentrations used.Conclusions:H2O2 comprehensively inhibits sperm qualities related to the capacity to fertilize oocyte, stimulates caspase activity, and inhibits cell survival.

Exposure to silver nanoparticles primes mast cells for enhanced activation through the high-affinity IgE receptor

Mast cells are a key effector cell in type I allergic reactions. It has been shown that environmental exposures such as diesel exhaust and heavy metals exacerbate mast cell degranulation and activation. Today, the use of engineered nanomaterials (ENMs) is rapidly expanding and silver nanoparticles (AgNP) are one of the mostly widely utilized ENMs, primarily for their antimicrobial properties, and are being incorporated into many consumer and biomedical products. We assessed whether pre-exposure of bone marrow-derived mast cells (BMMCs) to 20 nm AgNPs enhanced degranulation and activation to an allergen (dinitrophenol-conjugated human serum albumin) by measuring β-hexosaminidase release, LTB4 and IL-6 production. In addition, we assessed reactive oxygen species (ROS) generation, cell oxidative stress and toxicity as well as total and individual protein tyrosine phosphorylation (p-Tyr). We found that pre-exposure of BMMCs to AgNPs results in exacerbated allergen-mediated mast cell degranulation, LTB4 production and IL-6 release. Exposure of BMMCs to AgNPs exacerbated allergen-induced ROS generation, however, this was not associated with oxidative stress nor cell death. Finally, pre-exposure to AgNPs enhanced allergen-mediated global p-Tyr as well as individual proteins including Syk, PLCγ and LAT. Our findings indicate that pre-exposure to AgNPs exacerbates mast cell allergen-mediated phosphorylation of FcεR1-linked tyrosine kinases and ROS generation resulting in amplified early and late-phase responses. These findings suggest that exposure to AgNPs has the potential to prime mast cells to allergic immune responses, which could be of particular concern to atopic populations as the use of AgNPs in consumer and biomedical products rapidly increases.

Peptide-based NTA(Ni)-nanodiscs for studying membrane enhanced FGFR1 kinase activities.

Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of fibroblast growth factor receptors (FGFRs), and in the recruitment and activation of downstream intracellular signaling pathways. Biophysical and biochemical investigations of FGFR kinase domains in membrane environments offer key insights into phosphorylation mechanisms. Hence, we constructed nickel chelating nanodiscs based on a 22-residue peptide. The spontaneous anchoring of N-terminal His6-tagged FGFR1c kinase domain (FGFR1K) onto peptide nanodiscs grants FGFR1K orientations occurring on native plasma membranes. Following membrane incorporation, the autophosphorylation of FGFR1K, as exemplified by Y653 and Y654 in the A-loop and the total tyrosine phosphorylation, increase significantly. This in vitro reconstitution system may be applicable to studies of other membrane associated phenomena.

Regulation of boar sperm functionality by the nitric oxide synthase/nitric oxide system

PurposeNitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects.MethodsTwo main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media.ResultsOur results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes.ConclusionsThese findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.

PRMT1 Mediated FLT3 Methylation As a Therapeutic Vulnerability in FLT3-ITD+ AML

The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients.PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p<0.001). PRMT1-KD induced apoptosis was also more evident in cord blood (CB) CD34+ cells expressing FLT3-ITD (ShCtrl 19.4±1.3%, ShPRMT1 45.2±2.5%, p<0.001) relative to that of FLT3-WT (ShCtrl 27.47±1.8%, ShPRMT1 36.9±1.6%, p=0.0167). In 293T cells ectopically overexpressing FLT3-WT or FLT3-ITD, co-immunoprecipitation (co-IP) indicated greater interaction between PRMT1 and FLT3-ITD. Through RNA-Seq profiling two AML lines (MV4-11, OCI-AML3) plus one FLT3-ITD transduced CB CD34+ cells with PRMT1-KD, we obtained a differentially-regulated gene set as a “PRMT1 signature”. This signature was enriched in FLT3-ITD+ AML relative to FLT3-WT AML according to ssGSEA analysis using two AML datasets (GSE14468, GSE10358), indicating PRMT1 may cooperate with FLT3-ITD regulating AML maintenance.Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p<0.001, n=6). MS023 had little effect on long-term in vivo engraftment of CD34+ from a human CB specimen (vehicle 76.3±5.9%, MS023 72.2±3.4%, p=0.21, n=5).In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. DisclosuresKhaled:Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.

Enhanced Expression of Beta-Catenin and Axl Receptor Tyrosine Kinase (RTK) in Chronic Lymphocytic Leukemia (CLL) B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Leukemic Cell Drug Resistance

IntroductionCLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance.MethodsPurified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture.ResultsWe observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture.ConclusionsHere we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. [Display omitted] DisclosuresWarner:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Abstract 2144: Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance

Abstract Our study in CLL delineated a unique mechanism of how bone marrow stromal cells (BMSCs), may influence CLL B-cell signaling and drug resistance via modifying β-catenin and Axl status in leukemic cells. Purified primary CLL B-cells (n= 31) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=23) or CLL patients (n=12) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and real-time PCRs. We observed significant increases in expression of Axl at both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in co-cultured CLL B-cells. But, we did not see any significant change in β-catenin or Axl expression in BMSCs co-cultured with CLL B-cells. The CLL B-cells from co-culture had an upregulation in downstream P-ERK-1/2 but no change in P-AKT(Ser473). High nuclear active-β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β(Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β(Ser9) suggests inactivation of GSK-3β and β-catenin accumulation in co-cultured CLL B-cells. Additionally, we determined the phosphorylation status of Axl in CLL B-cells in co-culture compared with cultured alone. There was no change either at Y702 or total tyrosine phosphorylation in CLL B cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of Axl inhibitor TP-0903 on CLL B-cells in co-culture vs cultured alone. Interestingly, both Axl and β-catenin expression levels were further upregulated in CLL B-cells exposed to TP-0903 compared to CLL B-cells without TP-0903 in co-culture, indicating possible roles for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance. However, TP-0903 decreased P-Axl(Y702) as well as an Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells even in presence of BMSCs. This was associated with induction of cell death and overrode BMSC mediated protection. Treatments of fludarabine or chlorambucil also led to increase in expressions of both β-catenin and Axl in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. Here we show that stroma mediated increased expression in both β-catenin and Axl likely contribute to the survival and drug resistance of B-cells from patients with CLL. Understanding their role in drug metabolism is underway. Citation Format: Sutapa Sinha, Charla Secreto, Justin Boysen, Steven L. Warner, David J. Bearss, Asish Ghosh, Neil E. Kay. Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2144.

Treatment of Donor Rat Hearts Prior to Transplantation with FLIP (FADD-Like Interleukin Beta-Converting Enzyme (FLICE)-Like Inhibitory Protein) in Cardioplegic Solution Decreased Apoptosis at Thirty Minutes Post-transplantation and Decreased Total Tyrosine Phosphorylation Levels

BackgroundHeart transplantation is a therapeutic option for patients with severe coronary artery disease or heart failure. One of the difficulties to overcome is the apoptosis of cardiomyocytes in the donor organ. To prevent apoptosis in the donor organ, we developed a fusion protein containing FLIP (FADD-like interleukin beta-converting enzyme (FLICE)-like inhibitory protein) to inhibit caspase-8.Material/MethodsWe linked the cDNA coding for the FLIP protein to the transduction domain of HIV (human immunodeficiency virus) to allow the protein to enter cells. The recombinant protein was used at two different concentrations, 3 nM and 30 nM, for treatment of the donor heart in rat transplantation experiments. After transplantation, apoptosis was measured by ELISA, and the levels of active caspase-3, caspase-8, Bid, and PUMA were determined by western blotting using specific antibodies.ResultsWe observed that treatment of the donor organ with a solution containing this protein reduced the apoptosis level in the donor organ after 30 minutes post-transplantation as measured by the total of apoptotic cells with ELISA assay, and caspase-8 and caspase-3 activation and decreased levels of BH3-only proteins such as Bid and PUMA. Furthermore, this treatment also reduced the total tyrosine phosphorylation levels, which may be a possible measurement of lower oxidative stress levels in cardiomyocytes.ConclusionsProtein FLIP solution reduced apoptosis at 30 minutes post-transplantation and decreased levels of several regulators of apoptosis.

Uterine flushings from women treated with levonorgestrel affect sperm functionality in vitro.

Levonorgestrel (LNG), a synthetic 19 nor-testosterone derivative, is widely used for emergency contraception. It is well known that LNG prevents ovulation only when given prior to the surge of serum luteinizing hormone (LH) during the periovulatory phase of the menstrual cycle. This observation suggests that LNG, given its contraceptive efficacy, has additional effects other than those affecting ovulation. In this study, we have evaluated the effects on human sperm functionality of uterine flushings (UF) obtained from women at day LH + 1 of a control cycle (CTR-LH + 1) and after receiving LNG (LNG-LH + 1) two days before the surge of LH. Human sperm from normozoospermic donors were incubated with UF and protein tyrosine phosphorylation, sperm motility, acrosome reaction as well as zona pellucida (ZP) binding capacity were assessed. A significant decrease in total motility and tyrosine phosphorylation accompanied by an increase on spontaneous acrosome reaction was observed when sperm were incubated in the presence of LNG-LH + 1. None of these effects were mimicked by purified glycodelin A (GdA). Moreover, the addition of UF obtained during the periovulatory phase from LNG-treated women or the presence of purified GdA significantly decreased sperm-ZP binding. The data were compatible with changes affecting sperm capacitation, motility and interaction with the ZP. These results may offer evidence on additional mechanisms of action of LNG as an emergency contraceptive.

Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling.

We recently reported that maternal antibiotic treatment (MAT) of mice in the last days of pregnancy and during lactation dramatically alters the density and composition of the gastrointestinal microbiota of their infants. MAT infants also exhibited enhanced susceptibility to a systemic viral infection and altered adaptive immune cell activation phenotype and function. CD8+ effector T cells from MAT infants consistently demonstrate an inability to sustain interferon gamma (IFN-γ) production in vivo following vaccinia virus infection and in vitro upon T cell receptor (TCR) stimulation. We hypothesize that T cells developing in infant mice with gastrointestinal microbiota dysbiosis and insufficient toll-like receptor (TLR) exposure alters immune responsiveness associated with intrinsic T cell defects in the TCR signaling pathway and compromised T cell effector function. To evaluate this, splenic T cells from day of life 15 MAT infant mice were stimulated in vitro with anti-CD3 and anti-CD28 antibodies prior to examining the expression of ZAP-70, phosphorylated ZAP-70, phospho-Erk-1/2, c-Rel, total protein tyrosine phosphorylation, and IFN-γ production. We determine that MAT infant CD8+ T cells fail to sustain total protein tyrosine phosphorylation and Erk1/2 activation. Lipopolysaccharide treatment in vitro and in vivo, partially restored IFN-γ production in MAT effector CD8+ T cells and reduced mortality typically observed in MAT mice following systemic viral infection. Our results demonstrate a surprising dependence on the gastrointestinal microbiome and TLR ligand stimulation toward shaping optimal CD8+ T cell function during infancy.

Sensitive Approaches for the Assay of the Global Protein Tyrosine Phosphorylation in Complex Samples Using a Mutated SH2 Domain.

Temporal tyrosine phosphorylation (pTyr) plays a crucial role in numerous cellular functions. The characterization of the tyrosine phosphorylation states of cells is of great interest for understanding the underlying mechanisms. In this study, we developed sensitive and cost-effective methods for the assay of the global protein tyrosine phosphorylation in complex samples by using a novel engineered pTyr binding protein, Src SH2 domain triple-point mutant (Trm-SH2). Taking the advantage of the pan-specific interaction of Trm-SH2 to pTyr, a high throughput approach was developed to determine the total protein tyrosine phosphorylation level in a sample. This method allowed the detection of 0.025 ng of tyrosine phosphorylated proteins. The Trm-SH2 was further exploited to develop a method to profile the global tyrosine phosphorylation state. When this approach was applied to analyze the tyrosine phosphoproteome upon stimulation, distinct patterns were observed. This approach is readily used in many research and clinical fields for the analysis of tyrosine phosphorylated proteins in complex samples, including classifying aberrant phosphotyrosine-dependent signaling in cancer.

Toxicological Assessment of CoO and La2O3 Metal Oxide Nanoparticles in Human Small Airway Epithelial Cells.

Cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles are 2 metal oxide nanoparticles with different redox potentials according to their semiconductor properties. By utilizing these two nanoparticles, this study sought to determine how metal oxide nanoparticle's mode of toxicological action is related to their physio-chemical properties in human small airway epithelial cells (SAEC). We investigated cellular toxicity, production of superoxide radicals and alterations in gene expression related to oxidative stress, and cellular death at 6 and 24 h following exposure to CoO and La2O3(administered doses: 0, 5, 25, and 50 µg/ml) nanoparticles. CoO nanoparticles induced gene expression related to oxidative stress at 6 h. After characterizing the nanoparticles, transmission electron microscope analysis showed SAEC engulfed CoO and La2O3nanoparticles. CoO nanoparticles were toxic after 6 and 24 h of exposure to 25.0 and 50.0 µg/ml administered doses, whereas, La2O3nanoparticles were toxic only after 24 h using the same administered doses. Based upon the Volumetric Centrifugation Methodin vivoSedimentation, Diffusion, and Dosimetry, the dose of CoO and La2O3nanoparticles delivered at 6 and 24 h were determined to be: CoO: 1.25, 6.25, and 12.5 µg/ml; La2O3: 5, 25, and 50 µg/ml and CoO: 4, 20, and 40 µg/ml; and La2O3: 5, 25, 50 µg/ml, respectively. CoO nanoparticles produced more superoxide radicals and caused greater stimulation of total tyrosine and threonine phosphorylation at both 6 and 24 h when compared with La2O3nanoparticles. Taken together, these data provide evidence that different toxicological modes of action were involved in CoO and La2O3metal oxide nanoparticle-induced cellular toxicity.

Use of Dominant-Negative/Substrate Trapping PTP Mutations to Search for PTP Interactors/Substrates.

Phosphorylation of proteins on tyrosine residues is the consequence of coordinated action of tyrosine kinases (TKs), and protein tyrosine phosphatases (PTPs). Together, they regulate intermolecular interactions, subcellular localization, and activity of a variety of proteins. The level of total protein-associated tyrosine phosphorylation in eukaryotic cells is only a small fraction of the total phosphorylation. PTPs, which have high specific activity compared to tyrosine kinases, play an important role in maintaining the tyrosine phosphorylation state of proteins and regulate signal transduction pathways and cellular responses. PTPs depend on specific invariant residues that enable binding to substrates phosphorylated at tyrosine and aid catalytic activity. Identification of PTP substrates has helped understand their role in distinct intracellular signaling pathways. Because of their high specific activity, the interaction between tyrosine phosphatases and their substrates is often very transient in the cellular context, and therefore identification of physiological substrates has been difficult. Single-site mutations in the enzymes stabilize interaction between the enzyme and its targets and have been used extensively to identify substrates. The mutations are either of the catalytic cysteine (Cys) residue or other invariant residues and have been classified as substrate-trapping mutants (STMs). These mutants often serve as dominant negatives that can inactivate effector functions of a specific PTP within cells. Considering their association with human disorders, inhibiting specific PTPs is important therapeutically. Since the catalytic domains are largely conserved, developing small-molecule inhibitors to a particular enzyme has proven difficult and therefore alternate strategies to block functions of individual enzymes are seriously being investigated. We provide a description of methods that will be useful to design strategies of using dominant-negative and substrate-trapping mutants for identifying novel interacting partners and substrates of PTPs.

Aberrant Activation of the PI3K/mTOR Pathway Promotes Resistance to Sorafenib in AML

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant MV4-11 and MOLM-13 cell lines by sustained treatment with the FLT3 inhibitor sorafenib. MV4-11 cells express only FLT3-ITD, while MOLM-13 cells express wild-type FLT3 and FLT3-ITD. Both cell lines are dependent on FLT3 activation as sorafenib, PKC-412, and AC220, but not imatinib, dasatinib, nilotinib or bosutinib, inhibit cell survival in both cell lines. After treatment with sorafenib for 90 days, we observed that both cell lines displayed resistance to sorafenib as well as to AC220 suggesting that sustained treatment with an FLT3 inhibitor results in acquired resistance to multiple FLT3 inhibitors.To test whether sorafenib was still effective in FLT3 inhibition, we treated sensitive and resistant cells with sorafenib or DMSO and then stimulated with FLT3 ligand (FL). While DMSO-treated resistant cells responded to FL as expected, sorafenib-treated cells displayed poor FLT3 activation, suggesting that sorafenib was still capable of inhibiting FLT3 activation in the resistant cells. Surprisingly we observed that the resistant cells treated with DMSO had a much more robust response to ligand in terms of FLT3 activation. Similar results were observed with AKT and ERK activation, as sorafenib-treated cells poorly responded to FL-stimulation. Furthermore, resistant cells treated with sorafenib could still form colonies similar to DMSO-treated cells indicating that these cells were no longer dependent on FLT3 activation, although sorafenib could partially block FLT3 activation.To determine whether secondary mutations occurred in FLT3, we sequenced the whole coding region using Sanger sequencing. Except for a mutation in the extracellular domain in all four cell lines no mutations were detected in the inhibitor binding site. Similar to Sanger sequencing, mass spectroscopic analysis of affinity-enriched FLT3 indicated no differences in the intracellular part of FLT3 between sensitive and resistant cells. Because we observed an unexpected activation of FLT3 in resistant cells stimulated with FL, we hypothesized that certain FLT3 residues remain hyper-tyrosine phosphorylated. To test that we used phospho-specific antibodies against known FLT3 tyrosine phosphorylation sites. Although we observed an increase in total FLT3 tyrosine phosphorylation, we were unable to identify a single site that was selectively hyper-phosphorylated. Instead, all sites remained slightly more phosphorylated compared to control cells.Gene expression analysis of mRNA from sensitive and resistant cell lines using ANOVA and significance analysis of microarrays (SAM) suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype. In addition to pathway enrichment, using a phospho-protein antibody array, we found that phosphorylation of the mTOR substrates S6K and AKT were selectively increased in resistant cells. We also observed an increase in STAT3 phosphorylation. Elevated STAT3 phosphorylation was probably due to the previously described upregulation of JAK3 expression in sorafenib-resistant AML. We also observed enrichment of an mTOR signature in AML blasts from eight patients with sorafenib-resistant AML (GSE35907) and in AML blasts from patients expressing FLT3-ITD compared to those lacking FLT3-ITD (525 samples, GSE14468).Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in the resistant cell lines. Treatment of cells with a higher concentration of gedatolisib did not alter the phosphorylation of other signaling proteins except for AKT and S6K which was detected by a phospho-specific antibody array. The array data was further verified with western blotting using phospho-specific antibodies against AKT, ERK1/2, p38 and S6K. These results suggest that gedatolisib efficiently blocks the downstream effectors PI3K/mTOR without affecting other signaling pathways.Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD dependent AML results in resistance to drugs targeting FLT3 and treatment with selective PI3K/mTOR inhibitors are likely to be effective in overcoming acquired resistance in AML patients. DisclosuresStegmaier:Novartis Pharmaceuticals: Consultancy.

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization.

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10(6) sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY-stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.

The iNOS/Src/FAK axis contributes to lithium chloride-mediated macrophage migration

Lithium chloride (LiCl) has long been used as a mood stabilizer for bipolar mood depression patients. However, its biological effects on immune cells are unclear yet. In this study, we observed that upon LiCl stimulation, the motility and the content of total protein tyrosine phosphorylation in RAW264.7 macrophages and murine peritoneal macrophages (PEMs) were significantly increased. The inhibition of LiCl-induced macrophage migration by PP2 (an inhibitor for Src family kinases (SFKs)) suggested the involvement of SFKs in this process. Interestingly, LiCl induced NF-kB activation, and while Src was greatly induced, the expression of its myeloid relatives (i.e. Lyn, Fgr, Hck) was almost unaltered in RAW264.7 cells. Knockdown of Src suppressed LiCl-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by siRNA-resistant Src. Consistent with Src and migration increment was iNOS-dependent in macrophages, markedly reduced Src expression, activity and cell migration were observed in iNOS-null PEMs treated with LiCl. Moreover, FAK knockdown suppressed LiCl-stimulated macrophage motility, suggesting the involvement of FAK in this process. Remarkably, similar increment of iNOS, Src, FAK Pi-Tyr861 and migration ability could also be detected in RAW264.7 treated with other GSK3β inhibitors (i.e. SB216763 and Kenpaullone). These results corroborate that through inhibition of GSK3β, the iNOS/Src/FAK axis occupies a critical role in macrophage locomotion in response to LiCl.

Progesterone significantly enhances the mobility of boar spermatozoa

Progesterone released from the cumulus cells of the oocyte causes a number of physiological responses in human sperm cells including hyperactivation, acrosome reaction and chemotaxis. We employed a validated sperm mobility assay, which involves measuring the ability of sperm to penetrate an inert cell separation solution over time, to assess the ability of progesterone to enhance the mobility of boar spermatozoa. Cells maximally penetrate the solution over 50 minutes. 100nM progesterone significantly (P = 0.01) increased the mobility of non-capacitated sperm cells causing a doubling in the rate at which the cells penetrated through the cell separation solution (control half maximal penetration rate (Km) = 18.0±2.2; +100nM progesterone Km = 8.8±0.8min). Similarly, capacitated cells penetrated at a rate (Km = 19.2±3.0 min) not significantly different from non-capacitated cells and 100nM progesterone also significantly increased the rate of penetration of capacitated cells (Km = 9.5±1.0 min, P<0.05). The T-type voltage gated calcium channel blocker mibefradil (30mM) significantly inhibited both the control and progesterone enhanced mobility in non-capacitated and capacitated sperm. Only capacitated cells showed a significant increase in the acrosome reaction in response to 100nM progesterone (control non-reacted = 75±4%, +100nM progesterone non- reacted = 47±10%). Western blot analysis confirmed that there was an increase in the total protein tyrosine phosphorylation levels in capacitated cells. In conclusion, we have demonstrated that 100nM progesterone accelerates the mobility of boar sperm cells through an inert cell separation solution in an extracellular calcium dependent manner.