ALBERT

Description: Abstract 1036 Aims: Previous reports from this lab demonstrated importance of nitric oxide (NO) in augmenting neutrophils (PMNs) free radical generation. To explore further, present study investigated interaction of nitric oxide synthase (NOS) with Rac2 in resting and PMA activated human PMNs, and also nitration and nitrosylation of BSA following phagocytosis of coated beads. Results: Rac2 and iNOS interaction was evident in the cytosolic fraction of resting cells, which was augmented significantly in the membrane fraction of PMA treated PMNs. Nitration/nitrosylation of BSA, implicated functional importance of iNOS, and interaction of NO with NADPH-oxidase (NOX) and myeloperoxidase (MPO) derived species, as it was reduced significantly by the inhibitors of NOX (VAS), MPO (ABAH) and iNOS (1400W). Moreover, studies to assess the molecular characteristics and sub-cellular distribution of iNOS using standard methodologies, demonstrated iNOS transcript (RT-PCR, Real time PCR) and protein (immunoprecipitation and WB) in PMNs. iNOS protein was distributed in the granules, mitochondria, cytosol, nucleus and plasma membrane (immuno-electron microscopy and confocal microscopy) of human PMNs. Molecular characteristics, sub-cellular distribution of iNOS and its interaction with Rac2 are herein shown for the first time in human neutrophils. This study also elucidates the functional importance of iNOS-Rac2 interaction and highlighted the role of iNOS in nitration and nitrosylation of BSA following phagocytosis. Conclusions: Altogether these results suggest the functional importance of iNOS-Rac2 interaction in the nitration and S-nitrosylation of phagocytosed pathogens. Association of iNOS with Rac2 and its migration to the membrane in activated PMNs (A) Total cell lysate of human PMNs (1 × 108 cells) were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-iNOS (Lane 1) or anti-nNOS antibody (Lane 4). Lanes 2 and 5 – IP with rabbit IgG (negative control for anti-Rac2). iNOS recombinant protein (Lane 3) and rat brain homogenate protein (Lane 6) were used as positive controls for iNOS (130 kDa) and nNOS (160 kDa) respectively. Lanes 7 and 8 were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-Rac2. Inducible NOS and Rac2 interaction in the cytosol and membrane fractions of resting, PMA and NSC23766 with PMA stimulated cells. Both cytosol and membrane fractions were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-Rac2 (B) and anti-iNOS (C). The data represent 5 independent experiments, and are presented as Mean ± SEM (For Rac2, *** p

Description: Chronic myeloid leukemia (CML), a myeloproliferative disorder, characterized by sustained neutrophilia and constitutive BCR-ABL tyrosine kinase activity. Constitutive expression of the BCR-ABL kinase and elevated reactive oxygen species (ROS) levels through mitochondria and NADPH oxidase 4 (NOX4) activation leads to genomic instability and enhanced cell survival. Nitric oxide (NO), a signaling molecule has been associated with hematopoesis and suppression of NOS activity may induce profound changes in hematopoietic stem cells/progenitor cells. NO addition or iNOS transfection in K562 cells (BCR-ABL+) altered genes expression involved in the iron metabolism, inhibited cell proliferation and enhanced apoptosis, which were reversed by addition of exogenous iron. Moreover, anti-cancer effect of farnesyltransferase inhibitor in these cells was also mediated by NO production/iNOS induction. The present study investigates status and regulation of NO/iNOS in neutrophils from CML patients.The present study was undertaken to explore NO generation/iNOS expression and its regulation in circulating neutrophils so as to access the role NO/iNOS in CML pathology. All CML patients (Drug/treatment naïve, n=70; imatinib responders, n=62; imatinib resistant, n=25) included in this study were diagnosed in chronic phase. The study protocol was approved by the ethical committees of CSIR-CDRI and KGMU, Lucknow and was conducted in accordance with the declaration of Helsinki. Total nitrite level in neutrophils (PMNs) was assessed by Griess reagent. Nitric oxide, Superoxide, ROS/RNS and mitochondrial ROS generation was assessed by DAF-2DA, DHE, DCF-DA and Mitosox Red respectively. H2O2was measured by Amplex red assay kit. Expression of iNOS gene was evaluated by a SYBR green real-time RT-PCR and Western blot. Binding of NF-kB (p50 and p65 subunit) to iNOS promoter was analysed by CHIP assay. NF-kB (p50 and p65 subunit) and procaspase-3 glutathionylation was assessed by Immunoprecipitation followed by Western blot. Findings in CML patients were further validated using in vitro experiments on K562 cells. Statistical analysis were performed by one way ANOVA test followed by Newman-Keul’s post hoc analysis using the Graph Pad prism software. PMNs total nitrite, NO level and iNOS expression in drug naïve and imatinib resistant patients were significantly less as compared to healthy subjects. However, significant recovery in all the parameters was observed in imatinib responsive patients. Superoxide, ROS/RNS, mitochondrial ROS generation as well as H2O2 level was significantly more in drug naïve and imatinib resistant patients and it was attenuated significantly in imatinib responsive patient’s PMNs. In vitro treatment of K562 cells with Imatinib (2µM) also showed augmented NO generation and iNOS expression, while superoxide, ROS/RNS and mitochondrial ROS generation was decreased. To decipher the molecular mechanisms underlying the modulation of iNOS in BCR-ABL+ cells, we examined binding of NF-κB to iNOS promoter/enhancer and protein S-glutathionylation. Binding of NF-κB (p50 and p65 subunits) to iNOS promoter/enhancer was less in BCR-ABL positive cells, while it was augmented following treatment with imatinib. Moreover, glutathionylation of p50, p65 and procaspase-3 was more in drug naïve as well as in imatinib resistant CML patients PMNs, while it was comparable to healthy subjects in imatinib responders CML patients PMNs. Glutathionylation of NF-κB (p50 and p65 subunit) and procaspase-3 was also attenuated in imatinib treated K562 cells. The results obtained suggest that reduced NO generation/iNOS expression in BCR-ABL positive cells was due to the S-glutathionylation of NF-κB, which decrease it’s binding to iNOS promoter. S-glutathionylation of procaspase-3 in CML however, inhibited apoptosis of BCR-ABL positive cells. The study thus highlights importance of S-glutathionylation as key regulators involved in the proliferation and apoptosis of BCR-ABL positive cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4325 Introduction: Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm characterized by clonal proliferation of tumor cells that arise from the hematopoietic stem/progenitor population within the bone marrow. Nitric oxide (NO), a signaling molecule regulates hematopoietic stem and progenitor cells maturation by altering cell-cell adhesions, lineage-specific growth factors and actin confirmation (Michurina, et al, 2004). Moreover, suppression of NOS activity has profound effect on hematopoietic stem cells/progenitor cells, by increasing their self renewal or survival potential. Serum nitrite/nitrate level and expression of NOS is found to be more in AML patients (Brabdao et al, 2001). NO was also found to have cytotoxic potential in myeloid leukemia cells (Tsumori et al, 2002). Previous study from this lab has demonstrated that NO donor DETA NONOate in lower concentrations enhanced proliferation of HL-60 cells, while higher concentrations induced cytostasis, mitochondrial membrane potential loss and apoptosis (Kumar et al., 2010). Aim: The present study was undertaken to assess the circulating levels of NO metabolite, nitrite and NOS expressions in the circulating neutrophils and their precursor cells so as to assess the role of NO/NOS in AML pathology. Patients and Methods: Blood and bone marrow was collected from AML patients (mean age 33+10 years) after their informed consent. French American British criteria were used to diagnose AML pateints, which was based on the presence of 20% or more myeloblasts in bone marrow/blood smears. Twenty patients of AML were included and the study was approved by the Institutional ethics committee. Neutrophils (PMNs) and their precursor cells were isolated by Percoll density gradient and purity was confirmed by CD11b, CD15, CD16 labeling and by Giemsa staining. Total nitrite level in plasma and PMNs was assessed by Griess reagent and NOS expression was analysed by Real time RT-PCR. Results: Total nitrite level was less in AML patient's plasma and PMNs compared to healthy control (Figure 1A). Expression of both nNOS and iNOS was also less in AML patient's PMNs (Figure 1B). As AML is a disease of progenitor cells, further we have analysed nitrite level and NOS expression in PMNs progenitor cells {myeloblasts (MBs), promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), band cells (BCs) and segmented neutrophil (SNs)}. PMNs nitrite content and n/iNOS expression was less in all the progenitor cells compared to healthy control progenitor cells (Figure 2). In vitro results also showed that NOS expression was augmented following differentiation of HL60 cells in neutrophils. Conclusions: These observations indicate that NOS expression was significantly reduced in PMNs of AML patients. Further studies will be undertaken to assess the effect of NO modulators on the cell differentiation and apoptosis in AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Background Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one β-CA and two γ-CAs. Results One of the putative γ-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-γ-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. Conclusions This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized γ-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.