Biochemical Characterization and Gene Expression Profiling of Cytarabine Treated Stromal Fibroblasts Reveal Regulatory Mechanisms Affecting Hyaluronan and Heparan Sulfate Proteoglycans.

High dose chemotherapy and radiation, as applied in stem cell transplantation procedures, have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. However, the molecular mechanisms involved are far from elucidated yet. As bone marrow stromal glycosaminoglycans (GAGs) play an important role in various regulatory steps in hematopoiesis, we examined the effect of chemotherapy on the synthesis and composition of GAGs and gene expression profiles of the hematopoiesis supportive M210B4 fibroblastic stromal cell line. To this end GAGs were metabolically labeled with ³H-glucosamine and ³⁵S-sulfate, and analyzed by conventional biochemical techniques.

Cytarabine treatment resulted in a pronounced increase (1.6±0.3-fold increase) in hyaluronan (HA) and a reduction in the content (47±7% reduction) and in the extent of sulfation (³H-glucosamine to ³⁵S-sulfate ratio increased from 2,1 versus 2,6) of heparan sulfate proteoglycans (HSPG). Gene expression analysis showed a corresponding increase in hyaluronan synthase 1 (Has 1) gene expression and a decrease in hyaluronidase 2 (Hyal 2) gene expression. The decrease in HSPG was found to correspond with downregulation of the Exostosin 1 (Ext 1) gene, encoding a bifunctional glycosyltransferase, required for the biosynthesis of HS. Finally, there was a trend towards decreased expression of the N-deacetylase/N-sulfotransferase-1 (Ndst1) gene, which might explain the decrease in sulfation of HSPG after cytarabine treatment, as deacetylation is required for sulfation of HSPG to occur.

The functional consequence of the decrease in HSPG was investigated by Stromal Derived Factor-1α (SDF-1α) binding. SDF-1α was added to either untreated or treated M210B4 cells and allowed to bind for 90 minutes. After washing, bound SDF-1α was quantified by staining with the anti-SDF-1α antibody K15C. Cytarabine treatment resulted in a 31.1 ± 11.8% decline in K15C binding, indicating a decline in GAG-mediated binding of SDF-1α (n=6, Mann Whitney test, p<0.05). In conclusion, cytarabine treatment alters the expression of genes involved in GAG synthesis and degradation, thereby affecting the amount and structure of stromal GAGs. Accordingly, the chemokine-binding capacity of HSPG was negatively affected. In view of the critical role of SDF-1α in migration of stem cells and its presumed participation in the creation of the stem cell niche, our results suggest that chemotherapy-induced changes in stromal GAGs might affect homing and hematopoiesis. In addition, because of known functional capacities of HA, malignant hematopoiesis as well as tumor growth and invasion may be promoted.