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Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in human plasma. There are many cells and constitutions in human that can synthesis and excrete GPI-PLD. In these loci, bone marrow, hepar and pancreatic gland are the most important organs^[1-3]. Under some specific circumstances, only GPI-PLD can hydrolyze and release proteins from their GPI anchors on the plasma membrane of cells^[4]. The GPI-anchored proteins are functionally diverse and include cell-adhesion molecules, ectoenzymes, cell surface receptors, lymphocyte differentiation antigens, etc. These proteins have many functions involving adhesion, migration, regulation growth of cells. For instance, CD24 expressed on human white blood cells can regulate the adhesion and migration of these cells^[5-6]. So we speculated that GPI-PLD might have some effect on adhesion ability of leukemic cells by changing some GPI-anchored protein such as CD24. However, litter is known about the effect of GPI-PLD on the adhesion ability of myeloid leukemic cells through screening Medline and CNKI databases. This report was aimed to investigate the adhesion rate of bone marrow mononuclear cells (BMMNCs) separated from myeloid leukemia patients to fibronectin and CD24 expression after GPI-PLD activity being inhibited by 1,10-phenanthroline, a specific inhibitor for GPI-PLD in vitro.

An increasing number of cell surface proteins have been found to be anchored to the plasma membrane by GPI. These proteins are called GPI-anchored proteins. GPI-anchored proteins are functionally diverse and include cell-adhesion molecules, ectoenzymes, cell surface receptors, differentiation antigens. On the plasma membranes of human hematopoietic cells, approximately 10% of the antigens with defined CD expression pattern are GPI-anchored proteins. GPI-PLD is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. There are many evidences for the release of GPI-anchored proteins by intracellular GPI-PLD^[9]. GPI-PLD activity or expression may have effect on hematopoietic tumor cells adhesion and migration because some GPI-anchored proteins such as CD24 expressed on hematopoietic cells are also cell-adhesion molecules that facilitate tumor cells extramedullary infiltration^[10-11]. Therefore, it is significant to investigate GPI-PLD for cell adhesion, growth, apoptosis and differentiation. It is well known that many cells and constitutions in human such as liver, brain, pancreas, keratose cells, bone marrow stroma cells and hematopoietic cell lines express GPI-PLD mRNA, but most of it comes from liver, pancreas and bone marrow. Many studies indicate that the mount of GPI-PLD mRNA is correlated to the malignant phenotype of cells^[5]. The level of GPI-PLD expression in human ovarian cancer cells and epithelioma cells is higher than that in homologous normal cells. In the same time, a lot of tumor marks in cancer cells such as urokinaseis plasminogen activation receptor is generated. Therefore, GPI-PLD may take part in cancer infiltration and metastasis. Xie et al ^[3] discovered that human bone marrow cell lines K562, HL60, PLB-985, U937, and THP-1 express GPI-PLD. Our former experiment indicated that GPI-PLD activity and mRNA expression in BMMNCs separated from myeloid leukemia patients are higher than that from normal persons. So BMMNCs separated from myeloid leukemia patients were selected as research objects. The GPI-PLD activity could be decreased from (42.08±7.21)% to (5.4±2.96)% after BMMNCs were incubated with 1 mmol/L 1,10-phenanthroline for 5 hours in vitro and the adhesion rate increased from (49.78±26.73)% to (61.19±29.14)%. The difference was statistically significant (P < 0.01). These results illustrate that GPI-PLD activity is correlated to the adhesion ability of BMMNCs separated from myeloid leukemia patients. Decreasing GPI-PLD activity can increase the adhesion ability of BMMNCs separated from myeloid leukemia patients. The mechanism may be GPI-anchored adhesion molecules such as CD24 and CD87 increasing because of GPI-PLD activity decreasing. CD24 is a small heavily glycosylated glycosylphosphatidylinositol-linked cell surface protein, which is expressed in hematological malignancies as well as in a large variety of solid tumors such as non-small cell lung cancer, breast cancer, liver cancer, ovarian cancer^[10-13]. CD24 regulates cell adhesion by interaction with activated platelet and P-selectin on endotheliocyte^[14]. Many studies have revealed a highly significant association of increased cytoplasmic CD24 expression with shortened patient survival^[15]. The interaction of CD24 on tumor cells and P-selectin on activated platelet and endotheliocyte may facilitate cancer metastasis. Inhibiting GPI-PLD activity in BMMNCs promotes cells adhesion ability and CD24 expression. This may be due to CD24 promoting adhesion of cells with fibronectin. In order to explore the effect of GPI-PLD activity on leukemic cell adhesion ability, we inhibited GPI-PLD activity in vitro by 1,10-phenanthroline, a special inhibitor for GPI-PLD. The adhesion rate of BMMNCs to fibronectin and GPI-PLD activity were measured after GPI-PLD activity was inhibited by 1,10-phenanthroline and incubated by equal volume phosphate buffered saline. The results revealed that the adhesion rate of BMMNCs to fibronectin was increased significantly by inhibiting GPI-PLD activity. We think the increased adhesion ability of BMMNCs to fibronectin may due to increased cell adhesion molecules that expressed on BMMNCs because GPI-PLD activity was inhibited. So the CD24 expression was measured with Immunohistochemistry method. The result was in coincidence with our speculation. CD24 expression was increased after GPI-PLD activity had been inhibited. In summary, on the basis of our studies, the adhesion ability of BMMNCs separated from myeloid leukemia patients can be promoted by inhibiting GPI-PLD activity. Therefore, we have reason to speculate that GPI-PLD activity may be related to the prognosis and extramedullary infiltration of leukemia. But we still do not know if we can lower the infiltration ability of tumor cells and regulation the homing of hematopoietic stem cells by alteration GPI-PLD activity.

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