Liat Nadav

Heparanase mediates cell adhesion independent of its enzymatic activity

Heparanase is an endo‐β‐D‐glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanasemediated cell adhesion to extracellular matrix (ECM) results in integrin‐dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface‐bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM‐degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.— Goldshmidt, O., Zcharia, E., Cohen, M., Aingorn, H., Cohen, I., Nadav, L., Katz, B.‐Z., Geiger, B., Vlodavsky, I. Heparanase mediates cell adhesion independent of its enzymatic activity. FASEB J. 17, 1015–1025 (2003)

Heparanase expression in human leukemias is restricted to acute myeloid leukemias

OBJECTIVE Matrix metalloproteinases and an endo-beta-D-glucuronidase (heparanase) are enzymes that degrade the protein and carbohydrate constituents of basement membranes, thereby facilitating transendothelial migration of blood-borne cells. Heparanase activity was found to correlate with the metastatic potential of solid tumors. We evaluated heparanase expression, at the levels of gene and protein expression and activity in a variety of leukemias, and compared it with normal hematopoietic cells. MATERIALS AND METHODS Heparanase expression was evaluated in leukocytes isolated from peripheral blood of 71 patients with myeloid and lymphoid leukemias, or non-Hodgkins lymphoma. Analysis was performed at two levels: heparanase RNA was determined by reverse transcriptase polymerase chain reaction, and heparanase protein was evaluated by immunocytochemistry and flow cytometry. RESULTS In eight peripheral blood samples from normal donors, heparanase RNA was detected, and protein was found within the cytoplasm of granulocytes. In mononuclear cells derived from various leukemias, heparanase RNA was expressed in 14 of 15 acute myeloid leukemia (AML) samples. In contrast, cells derived from all 33 chronic lymphoblastic leukemia, all 7 non-Hodgkins lymphoma, 7 of 8 chronic myeloid leukemia, and 6 of 8 acute lymphoblastic leukemia patients showed no detectable expression of the heparanase RNA. Heparanase protein was detected primarily within the cytoplasm of AML cells, indicating that the enzyme is produced and stored within the cytoplasm of myeloid cells, with limited expression on the cell surface. CONCLUSION We propose that heparanase expression is associated with the myeloid lineage and may serve as an independent marker to support the identification of AMLs.

Diverse niches within multiple myeloma bone marrow aspirates affect plasma cell enumeration

A basic criterion for the diagnosis of multiple myeloma is plasma cell enumeration within the bone marrow (BM). This report showed that flow cytometry under‐estimated the number of plasma cells in BM aspirates by an average of 60%, compared with morphological evaluation. The discrepancy was partially because BM smears contain cells associated with the lipid‐enriched spicules. In contrast, flow cytometry is performed on the BM fluid, which is depleted of the lipid‐adhesive plasma cells. This discrepancy may point to different plasma cell subpopulations associated with diverse niches within the BM.

The Generation and Regulation of Functional Diversity of Malignant Plasma Cells

Cellular diversity, which is a hallmark of malignancy, can be generated by both genetic and nongenetic mechanisms. We describe here variability in the adhesive and migratory behavior of malignant plasma cell populations, including multiple myeloma-derived lines and primary patient samples. Examination of the plasma cell lines ARH-77, CAG, and AKR revealed two distinct subpopulations of cells, one displaying highly adhesive properties (type A) and the other consisting of poorly adhesive, floating cells (type F). In the ARH-77 cell line, type A cells attach better to fibronectin and to human bone fragments and form paxillin-rich focal adhesions, whereas type F cells are highly motile and exert integrin-dependent bone marrow homing capacity in nonobese diabetic/severe combined immunodeficient mice. Flow cytometry indicated that type A cells express significantly higher levels of CD45 and CD56 and lower levels of CD138 compared with type F cells. Interestingly, culturing of either type A or type F cells under nonselective conditions resulted in the development of mixed cell population similar to the parental ARH-77 cells. Analysis of bone marrow aspirates of multiple myeloma patients revealed that spicules within the aspirates are enriched with type A-like cells. Nonadherent cells within the aspirate fluids express a marker profile similar to type F cells. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells that display distinct properties. Diverse subpopulations of malignant plasma cells may play distinct roles in the different biological and clinical manifestations of plasma cell dyscrasias, including bone dissemination and selective adhesion to bone marrow compartments.

Tumorigenic potential and disease manifestations of malignant B-cell variants differing in their fibronectin adhesiveness

OBJECTIVE Microenvironmental interactions of malignant B cells can modulate various in vitro physiological responses, including proliferation, migration, apoptosis, and drug resistance. Disease manifestations of human malignant B-cell variants, isolated based on their differential interactions with fibronectin, were examined in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. MATERIALS AND METHODS Disease manifestations were assessed by pathological examinations and skeletal imaging of NOD/SCID mice injected with malignant B-cell variants. Dissemination patterns were analyzed by whole-body real-time imaging of mice injected with fluorescence-labeled malignant cells. RESULTS Initial dissemination patterns and dynamics of both high (type A) and low (type F)-adherent variants, following intravenous inoculation, were similar. Both cell types reached the spleen and liver within 30 minutes after injection, then increasingly accumulated within the bone marrow. Mice injected with type-A cells developed multiple myeloma-like disease within the bone marrow, with multiple lytic bone lesions. In contrast, type-F cells displayed low tumorigenic capacity in spite of their efficient homing to the bone marrow niche. In addition, type-A cells grew as extramedullary tumors in some of the intravenous-inoculated mice, and formed solid tumors following subcutaneous injection. Both cell variants retained their characteristics surface markers following in vivo outgrowth as tumors, indicating that at least some of their properties are relatively stable. CONCLUSION Data suggest that the differential tumorigenicity of B-cell adhesive variants is attributable to the capacity of type-A cells to survive and proliferate within the bone marrow, rather than to different initial dissemination of the two cell populations.