A Glycomic Approach Towards Identification of Signature Molecules in CD34+ Haematopoietic Stem Cells from Umbilical Cord Blood.

Umbilical cord blood (UCB) is a powerful storehouse for normal CD34+ haematopoietic stem cells (HSCs), often used for allogeneic bone marrow (BM) transplantation in malignant and non-malignant diseases. The glycomic especially the sialoglycomic aspect of these HSCs has been unravelled in this study. Cell surface expression of the glycans with the related enzymatic activities has been compared with the BM of childhood acute lymphoblastic leukaemia, a common BM-associated malignancy. An enhanced cell surface expression of α2,3-linked sialic acid, P- and E-selectins, and intercellular adhesion molecule along with reduced expression of L-selectin distinguishes CD34+ HSCs of UCB from leukaemic samples. More importantly, high expression of O-acetylated sialoglycoproteins, a hallmark of lymphoblasts, is drastically reduced in the CD34+ HSCs of UCB and is substantiated by the low activity of sialylate-O-acetyltransferase and high sialidase activity. In contrast, a significant variation is evident in the expression of sialic acid, α2,6-linked sialic acids, and the sialyltransferase activity. Taken together, these studies indicate a few signature molecules, forming a unique glycomic template, which may be a potential indicator, reassuring the normal profile of these stem cells, to be used for future transplantation.

9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia.

Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac2-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac2-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH(+)SSC(lo)CD45(hi)Neu5,9Ac2 -GPs(lo)CD34(+)CD38(-)CD90(+)CD117(+)CD133(+)) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH(+)SSC(lo)CD45(lo)Neu5,9Ac2 -GPs(hi)CD34(+)CD38(+)CD90(-)CD117(-)CD133(-)) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy.

Mobilization of lymphoblasts from bone marrow to peripheral blood in childhood acute lymphoblastic leukaemia: role of 9-O-acetylated sialoglycoproteins.

Childhood acute lymphoblastic leukaemia is characterized by aberrant proliferation and accumulation of malignant lymphoblasts in bone marrow (BM), followed by their migration into circulation. An enhanced cell-surface expression of ALL-associated 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) was demonstrated. Present investigation reports a positive correlation between the increased density of Neu5,9Ac(2)-GPs on lymphoblasts and their mobilization from BM involving enhanced Neu5,9Ac(2) on CD45 demonstrating modulation of FAK and ERK molecules. In contrast, a small population of cells, identified as haematopoietic precursors, with comparatively lesser Neu5,9Ac(2)-GPs showed increased binding towards BM stroma. Thus, Neu5,9Ac(2)-GPs is a developmentally regulated oncofoetal antigen, whose up-regulation is imperative in the interaction between lymphoblasts and BM stroma, governing their mobilization into circulation.

O-acetylated sialic acids: multifaceted role in childhood acute lymphoblastic leukaemia.

Childhood acute lymphoblastic leukaemia (ALL), a malignant transformation of the lymphoblasts, is highly responsive to chemotherapy. However, due to certain inadequacy in detection of minimal residual disease (MRD), relapse is a common phenomenon. To address this question, the present review deals with the induction of an unique O-acetyl derivative of sialic acid on a few disease-associated glycoproteins and glycolipids at the onset of childhood ALL, a finding of our group in the last decade. This information has been successfully utilized for diagnosis and prognosis of the disease. Existing literature is included for comparison. Additionally, cell surface overexpression of 9-O-acetylated sialoglycoproteins and antibodies against them present in patients' sera aid the survival of the malignant lymphoblasts and suggest a multifaceted role played by these molecules. Taken together, monitoring these molecules helps not only in unravelling the biology of this paediatric malignancy but also in personalizing the treatment strategies for the betterment of the patient population.

High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status.

Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac(2)-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the V (max) of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.

Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation.

INTRODUCTION: Resistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated. MATERIALS METHODS AND RESULTS: Menadione synergized NaAsO(2) to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-L: -cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-kappaB upon NaAsO(2) and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC. CONCLUSION: This study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.

Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study.

BACKGROUND: Over expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Achatinin-H, a lectin, has selective affinity towards terminal 9-O-acetylated sialic acids-alpha2-6-Nacetylated galactosamine. Exploring this affinity, enhanced expression of OAcSGP was observed, at the onset of disease, followed by its decrease with chemotherapy and reappearance with relapse. In spite of treatment, patients retain the diseased cells referred to as minimal residual disease (MRD) responsible for relapse. Our aim was to select a suitable template by using the differential expression of OAcSGP along with other known CD antigens to monitor MRD in peripheral blood (PB) and bone marrow (BM) of Indian patients with B- or T-ALL during treatment and correlate it with the disease status. METHODS: A two-year longitudinal follow-up study was done with 109 patients from the onset of the disease till the end of chemotherapy, treated under MCP841protocol. Paired samples of PB (n = 1667) and BM (n = 999) were monitored by flow cytometry. Three templates selected for this investigation were OAcSGP+CD10+CD19+ or OAcSGP+CD34+CD19+ for B-ALL and OAcSGP+CD7+CD3+ for T-ALL. RESULTS: Using each template the level of MRD detection reached 0.01% for a patient in clinical remission (CR). 81.65% of the patients were in CR during these two years while the remaining relapsed. Failure in early clearance of lymphoblasts, as indicated by higher MRD, implied an elevated risk of relapse. Soaring MRD during the chemotherapeutic regimen predicted clinical relapse, at least a month before medical manifestation. Irrespective of B- or T-lineage ALL, the MRD in PB and BM correlated well. CONCLUSION: A range of MRD values can be predicted for the patients in CR, irrespective of their lineage, being 0.03 +/- 0.01% (PB) and 0.05 +/- 0.015% (BM). These patients may not be stated as normal with respect to the presence of MRD. Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival. Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

9-O-acetylated GD3 triggers programmed cell death in mature erythrocytes.

An acetylated modification of a tumor-associated ganglioside GD3 (9-O-AcGD3) is expressed in certain tumors and present during early stages of development in different tissues. However, the status and the role of 9-O-AcGD3 in the erythroid progenitor cells remain unexplored. Here, we report the level of 9-O-AcGD3 during erythropoiesis in bone marrow is down regulated during maturation. Signaling via 9-O-AcGD3 induces alteration of morphology and membrane characteristics of mature erythrocytes. This process also induces, a cell death program in these erythrocytes even in the absence of nucleus, mitochondria and other cell organelles sharing features of apoptosis in nucleated cells like membrane alterations, vesicularization, phosphatidyl serine exposure, activation of cysteine proteases like caspase-3. This is the first report of a programmed cell death pathway in mature erythrocytes, triggered by 9-O-AcGD3 contrary to their anti-apoptotic role in lymphoblasts, which suggests a cell specific role of this O-acetyl ester of GD3.

Comparative analysis of differential expression of sialic acids and adhesion molecules on mononuclear cells of bone marrow and peripheral blood in childhood acute lymphoblastic leukaemia at diagnosis and clinical remission.

Childhood acute lymphoblastic leukaemia (ALL) is characterized by the neoplasm of immature haematopoietic precursor cells (HPCs). We report significant differences between the expression of sialoglycoproteins and adhesion molecules on mononuclear cells (MNCs) of bone marrow (BM) and peripheral blood (PB) from individual children at diagnosis of the disease. Lymphoblasts in PB predominantly expressed 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs), sialic acid, alpha2-3 linked sialic acid, L- and P-selectins and vascular cell adhesion molecule -1 (VCAM-1) on their surface compared to BM, as determined with selective lectins and monoclonal antibodies (mAbs) by flow cytometric analysis. CD34+CD38+ cells present either in diagnostic PB or BM always showed enhanced expression of both alpha2-3 and alpha2-6 linked sialic acids, Neu5,9Ac2-GPs, L- and P-selectins and VCAM-1, compared to CD34+CD38- population, as confirmed by higher mean fluorescence intensity (MFI). Expression of ICAM-1 was reverse. However, MFI of Neu5,9Ac2-GPs was always higher both in CD34+CD38+ and CD34+CD38- population in PB compared to BM. Diverse trend of these cell surface macromolecules was observed during clinical remission. This is the first comparative study between PB and BM, where significant differential distribution of sialylated macromolecules and adhesion molecules was observed. Hence, supervising these cell surface macromolecules at various stages of treatment might help in minimal residual disease detection, identifying mobilization factor(s) and in isolation of normal HPCs for autologous BM transplantation.

Role of C-reactive protein in complement-mediated hemolysis in Malaria.

Human C-reactive protein (CRP) is a clinically important classical acute phase protein. Although CRP has been reported to bind with many nucleated cells, the direct binding of CRP to erythrocytes in diseases remains largely unexplored. The main focus of the present study was to investigate the binding of disease-specific CRP to erythrocytes of same patients. Distinct molecular variant of disease-specific CRP was affinity purified from sera of malaria patients (CRP(Mal)). This CRP showed strong binding with malaria erythrocytes (RBC(Mal)) as confirmed by flow cytometric analysis (FACS), enzyme-linked immunosorbent assays (ELISA), and radio binding assays. Calcium and phosphoryl choline (PC) were found to be essential for this interaction. A 2.3-fold increased binding of induced CRP to RBC(Mal) as compared to normal erythrocytes (RBC(N)) confirmed disease-specificity. Preincubation of RBC(Mal) with unconjugated CRP showed 3-5 fold inhibition. The association constant of CRP and RBC(Mal) was 4.7 x 10(6) cpm/microg with the corresponding number of receptors/cell being 4.3 x 10(5). The effector function of CRP(Mal) has been demonstrated by its potency to activate the complement pathway. An optimal dose of 10 microg/ml of CRP induced three-fold higher hemolysis of patient erythrocytes as compared to RBC(N). These studies provide direct evidence for an important phagocytic functional interaction of this acute-phase protein by triggering the CRP-complement pathway after the binding of CRP(Mal) with RBC(Mal). Hemolysis as triggered by this pathway may be one of the causative factors of anemia, a common clinical manifestation of this disease.