heparitin-sulfate and Leukemia--Myeloid

Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS). To this end, PG structure was altered by desulfation or enzymatic cleavage. PGs participated in adhesion of human HPC, as desulfation resulted in about 50% decline in adhesion to hBMS. Heparan sulfate proteoglycans (HSPGs) were found to be responsible by showing about 25% decline in adhesion after pre-incubation of HPC with heparin and about 15% decline in adhesion after enzymatic removal of HSPGs from hBMS. Furthermore, PGs were involved in binding cytokines. Both desulfation and enzymatic removal of stromal HSPGs increased release of megakaryocytopoiesis-inhibiting cytokines, that is, interleukin-8 (IL-8, 1.9-fold increase) and macrophage inflammatory protein-1alpha (MIP-1alpha, 1.4-fold increase). The megakaryocytic output of HPC grown in conditioned medium of desulfated stroma was decreased to 50% of the megakaryocytic output in CM of sulfated stroma. From these studies, it can be concluded that PGs in bone marrow, in particular HSPGs, are involved in binding HPC and megakaryocytopoiesis-inhibiting cytokines. Bone marrow stromal PGs thus reduce differentiation of HPC toward megakaryocytes.

Topics: Acute Disease; Antigens, CD34; Blood Proteins; Bone Marrow; Cell Adhesion; Cell Differentiation; Cells, Cultured; Chemokine CCL3; Chemokine CCL4; Culture Media, Conditioned; Eosinophil Major Basic Protein; Hematopoietic Stem Cells; Heparitin Sulfate; Humans; Interleukin-8; Leukemia, Myeloid; Lymphoma, Non-Hodgkin; Macrophage Inflammatory Proteins; Megakaryocytes; Proteoglycans; Stromal Cells

Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

Topics: Acute Disease; Adenoviridae; beta-Galactosidase; Capsid; Capsid Proteins; Cell Line; Cells, Cultured; Genetic Therapy; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Heparin; Heparitin Sulfate; Humans; Interleukin-3; Leukemia, Myeloid; Stem Cell Factor; Transfection

Conditioned medium from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix induces macrophage-like differentiation of fresh HL-60 cells. The active medium component is sensitive to protease treatment, indicating that it is a protein, but it is heat stable. Conditioned medium from HL-60 cells grown on protease-treated bone marrow matrix still contains the active component. Thus, it appears that the differentiation-inducing protein is produced by HL-60 cells and is not released from the bone marrow matrix. To identify this differentiation factor, RNA was isolated from HL-60 cells grown on bone marrow matrix and assayed by Northern analysis for expression of mRNA for human differentiation factor, tumor necrosis factor, and macrophage colony-stimulating factor, all inducers of monocyte/macrophage differentiation. Expression of differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor mRNA was not enhanced in HL-60 cells grown on matrix compared to cells grown on uncoated plastic flasks. Thus, the maturation factor does not appear to be differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor within the limits of detection of Northern analysis. Elution of the active conditioned medium fraction on a Sephacryl S-200 column revealed a molecular weight of approximately 40,000. The active protein eluted on a DEAE-cellulose ion-exchange column at an ionic strength of 0.3 M NaCl, indicating that it is fairly anionic. Thus, bone marrow matrix is able to induce HL-60 cells to produce a maturation-inducing 40 kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biological Factors; Blotting, Northern; Bone Marrow; Cell Differentiation; Chromatography, Gel; Chromatography, Ion Exchange; Culture Media; Extracellular Matrix; Heparitin Sulfate; Humans; Leukemia, Myeloid; Radioligand Assay; Tumor Cells, Cultured

The average glycosaminoglycan content in control spleens, expressed as uronic acid, was 0.23 +/- 0.02 mg/g of dry wt; the average glycosaminoglycans content in spleens of CML patients, expressed as uronic acid, was 0.91 +/- 0.23 mg/g of dry wt. In control and in leukemic spleens the same glycosaminoglycans were present, that is hyaluronic acid, heparan sulphate, dermatan sulphate, chondroitin-4-sulphate and chondroitin-6-sulphate. However, in leukemic spleens the normal quantitative relationship between these glycosaminoglycans was greatly modified; in fact in control spleens hyaluronic acid, heparan sulphate and the chondroitin sulphates were present in almost equal proportions, whereas in leukemic spleens the chondroitin sulphate group alone represented almost 9/10 of all the glycosaminoglycans. Since this proportion is weakly modified in leukemic spleens in which the number of myeloid cells has been notably reduced after chemotherapy, we may suppose that this phenomenon is due to the very marked modifications which take place in the micro-environment of the leukemic spleen.

Topics: Chondroitin Sulfates; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Leukemia, Myeloid; Spleen