A Heparan Sulfate-containing Fraction of Bone Marrow Stroma Induces Maturation of HL-60 Cells in Vitro

Constituents of the bone marrow microenvironment have the capacity to influence both normal and malignant hematopoietic cell behavior. For example, HL-60 human promyelocyte leukemia cells in vitro display a more mature phenotype when grown on a bone marrow stroma-derived matrix. To elucidate which component(s) of the stromal matrix is capable of modulating HL-60 cell phenotype, matrices were treated with a variety of chemicals and enzymes prior to being used in the differentiation assay. Treatment of matrices with collagenase, pronase, chondroitinase, or chloroform:methanol:ether could not abolish the differentiation-promoting activity of bone marrow stroma. In contrast, the activity was destroyed by alkali treatment (0.5 m NaOH for 18 h) or heparinase/heparitinase enzymes. Heparin added to cultures increased maturation of HL-60 cells as determined by esterase production, Fc rosette formation, and morphological appearance. Other stromal components such as laminin, fibronec-tin, collagen I, collagen IV, or chondroitin sulfate did not alter the HL-60 leukemia cell phenotype. Stroma-derived matrix material which labeled with [35S]sulfate and eluted on a DEAE ion-exchange column as a high ionic fraction in 1.5 m LiCl and 7.5% sodium dodecyl sulfate contained the active fraction. A heparan sulfate proteoglycan component isolated by polyacrylamide-agarose gel electrophoresis induced a more mature HL-60 phenotype, and digestion with heparinase/heparitinase in the presence of protease inhibitors abrogated the effects on HL-60 phenotype. We conclude that a heparan sulfate-associated fraction of the bone marrow matrix plays a key role in the regulation of leukemic cell maturation.