Elsevier

Clinical Immunology

Volume 122, Issue 2, February 2007, Pages 194-206
Clinical Immunology

Expression of human liver HSPGs on acute myeloid leukemia

https://doi.org/10.1016/j.clim.2006.08.017Get rights and content

Abstract

Heparan sulfate proteoglycans (HSPGs) play important biological roles in cell–matrix adhesion processes and are essential regulators of growth actions. The expression of the different HSPGs in itself is tightly regulated providing strict controls on the activities of the bound ligands. Human liver is a target for a number of pathogens, and HSPGs have been demonstrated in several cases to play a pivotal role in infectivity. Despite HSPGs important biological functions, little is known about its cell-specific distribution patterns. Human liver HSPG was isolated, and a specific monoclonal antibody (mAb) 1E4-1C2 was produced. Distribution of HSPG reactive to this mAb was studied in normal blood cells, hematopoietic cell lines and blood cells isolated from patients with various hematologic disorders using indirect immunofluorescence. There was no expression of molecules recognized by this mAb on lymphoid (Daudi, Jurkat, SupT-1) and monocytoid (U937) cell lines. Peripheral blood cells, normal bone marrow, together with leukocytes isolated from patients with acute lymphoblastic leukemia, chronic myelocytic leukemia, Hodgkin’s disease or Non-Hodgkin’s lymphoma, were also negative. In contrast, 1E4-1C2 showed significant positive results on human myeloid cell lines HL-60 and K562. Moreover, it is interesting that this mAb also recognized epitopes on leukocytes isolated from acute myeloblastic leukemia. These results suggest that malignancies of cells in myeloid lineage may cause expression of HSPGs that are detected by this specific mAb, making it a potential co-marker for the diagnosis of acute myeloid leukemia.

Introduction

Acute myeloid leukemia (AML) is a complex disease with considerable phenotypic and genotypic heterogeneity. Most cases of AML are sporadic, characterized by acquisition of somatic mutations in hematopoietic progenitors that confer a proliferative and/or survival advantage, impair hematopoietic differentiation and confer properties of limitless self-renewal [1]. A recent study suggests that the certain leukemia oncogenes such as the MLL–ENL fusion commandeer programs of self-renewal in committed progenitors and enforce phenotypical expression of markers associated with hematopoietic stem cells [2]. These may not only be valid targets for therapeutic intervention, but might also provide insights into approaches to confer properties of self-renewal to adult somatic cells for therapeutic benefit, such as tissue regeneration. AML is usually diagnosed by examination of a patient with a blood smear having a total white count from 1000/mm3 to > 200,000/mm3. Ten percent of patients, however, still have normal white blood count without circulating blasts. Bone marrow aspiration and biopsy also need to be performed for cytogenetic and immunophenotypic studies. Bone marrow in a patient with AML typically is hypercellular, with absent or a decreased level of megakaryocytes [3]. Cytogenetic studies are important prognostic indicators and are generally performed at the time of the initial bone marrow aspiration and biopsy [4]. Cytogenetic as well as molecular genetic studies are of major importance in diagnosing AML and defining the major AML subtype [4]. However, these tests require viable and dividing cells and can take up to 2 weeks for a complete analysis.

Classification and diagnosis of AML basically rely on the morphologic and cytochemical system proposed in 1976 [5], [6]. The World Health Organization has revised this classification system to include unique clinical and biological subgroups (M0–M7). Immunophenotyping, using monoclonal antibodies (mAbs) to identify myeloid- and lymphoid-associated antigens, has been very helpful in defining such subgroups. The percentage of positive reacting blasts should be greater than 20%, with one or more of myeloid-associated antigen, CD33 or CD13, to establish the presence of AML [7]. Individual antibodies that correlate well with the morphologic classification include CD34 and CD117 in subgroups M0 and M1. The expression of these surface antigens on myeloblasts, however, does not always agree with morphologic or cytogenetic staining features [8], [9]. Therefore, the use of additional mAbs that can identify other phenotypic markers might be of clinical importance. A majority of myeloid blast cells express differentiation markers asynchronously, and this unusual coexpression of normal differentiation antigens is common.

HSPGs interact with various extracellular matrix components, adhesion molecules, proteinase inhibitors, growth factors and transcription factors and control spatial distribution and bioavailability of these molecules. HSPGs in liver act as a critical receptor for apoE and are involved in lipid metabolism [10], [11] and are likely involved in some of the pathology associated with Alzheimer’s disease [12], [13]. Human liver is also a target of a number of pathogens, and heparan sulfate has been demonstrated in several cases to play a pivotal role in infectivity [14], [15], [16]. The liver is known to be a rich source of HSPGs. While heparin is intracellular and found in granulated cells [17], HSPGs are extracellular and commonly found on cell membranes, suggesting their importance as a receptor or coreceptor for various heparin-binding proteins [18]. Recently, human liver HSPG with an elevated sulfation level was isolated and purified [19]. Since HSPGs are involved in controlling the growth and differentiation of a variety of cell types, mAbs were prepared against this human HSPG and used as a tool to distinguish between hematopoietic cell types. We report that human liver HSPG is expressed on the other cell types, most interesting on certain malignant cells in myeloid lineage. We propose to apply a specific mAb prepared against this human HSPG as a tool to detect leukemic cells.

Section snippets

Cells, reagents and antibodies

HSPG D2 fraction, used as an antigen in this study, was isolated and purified from human liver [19]. Normal blood samples were buffy coats isolated from healthy normal humans. Hematopoietic cell lines (Daudi, Sup T-1, Jurkat, U937, HL-60 and K562) used in this study were kindly provided from Dr. Watchara Kasinrerk, Faculty of Associated Medical Sciences, Chiang Mai University. All cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS (Gibco, Grand Island, NY), 40 μg/ml

Results

HSPGs are an important group of molecules involved in many cellular processes especially those of growth and differentiation. A human liver HSPG was isolated, fractionated and purified to better understand its mechanism of action. Specific mAb was prepared against the highly sulfated D2 fraction (Fig. 1) using standard hybridoma technique [19]. Clones were characterized and used as a tool to study the expression of human liver HSPGs. The mAb 1E4-1C2 recognizes an HSPG having a molecular weight >

Discussion

In the current study, we isolated, fractionated and purified liver HSPG from normal healthy donor. The most highly sulfated HSPG fraction, D2, was selected since it contained an intact core protein and isolated amounts sufficient for immunization and screening. We prepared a set of mAbs reactive with the D2 HSPG fraction. Next, these mAbs were used to screen for epitopes on hematopoietic cells. The 1E4-1C2 mAb was found to be of interest since it was able to detect an epitope on cell lines in

Acknowledgments

This work was supported by Thailand Research Fund 2003 (grant number MRG4680128), IRPUS (grant number I4801001) and National Research Council (2549). The researcher would like to thank Dr. Weerasak Nawaravong, Faculty of Medicine, Chiang Mai University, for his kindly provided blood samples. This work was approved by the Committee of Research Ethics (number 283/2548).

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