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considered a model system for understanding the physiopathology of human leukemia , the multistage nature of leukemogenesis , and the molecular mechanisms of leukemia resistance

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin E1, used as positive control. Prostaglandin E1 added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias. Key terms: angiogenesis, chick embryo chorioallantoic membrane assay, Friend erythroleukemia, human endothelial cells Corresponding Author: Dr. Tiziana Punzi, Dept. of Anatomy, Histology and Forensic Medicine, University of Firenze, Viale Morgagni 85, 50134 Firenze, Italy; Phone: +39 055 410084; Fax: +39 055 4379500; E-mail: tizianapunzi@email.it Received: June 26, 2007. In Revised form: April 18, 2008. Accepted: May 5, 2008 INTRODUCTION Angiogenesis is a prerequisite for sustained growth of primary tumors and metastases. Angiogenesis is different from vasculogenesis as this latter is a biologic process by which new blood vessels originate from endothelial cell precursors, whereas in angiogenesis, new blood vessels originate from pre-existing blood vessels (Yancopoulos et al., 1998). Angiogenesis occurs both in physiologic and pathologic conditions, and tumor cells as well as normal growing cells produce and secrete several growth factors and signaling molecules involved in the control of angiogenesis. Among the signals controlling angiogenesis, prostaglandins play a major role. An example is represented by Prostaglandin E1 (PGE1), which induces angiogenesis by stimulating transcription of the genes coding for the Vascular Endothelial Growth Factor (VEGF) and of other pro-angiogenic factors, such as basic Fibroblast Growth Factor (bFGF) and Transforming Growth Factor-β (TGF-β) and interleukin 6 (IL6) (Francis, 2002). It has long been known that successful angiogenesis is required for sustained growth of virtually all solid tumors. Recent studies, however, have also demonstrated the importance of angiogenesis in normal hematopoietic cell development as well as in the pathophysiology of several hematologic malignancies including leukemias (Shanafelt and Kay, 2006). Among the different experimental models exploited to investigate leukemias, Friend erythroleukemia cells are PACINI ET AL. Biol Res 41, 2008, 109-117 110 considered a model system for understanding the physiopathology of human leukemia, the multistage nature of leukemogenesis, and the molecular mechanisms of leukemia resistance (Smadja-Joffe et al., 1975; Ney and D’Andrea, 2000). Nonetheless, the relationship between Friend erythroleukemia and angiogenesis has not yet been studied. Based upon these assumptions, we studied the angiogenic response induced by Friend erythroleukemia cells in two wellestablished models for angiogenesis investigation, i.e., the chick embryo Chorioallantoic Membrane (CAM) assay and Human Umbilical Vein Endothelial Cells (HUVEC) cultures. Given the fundamental role of prostaglandins in the control of angiogenesis, one of these signaling molecules (PGE1) was used as control. The use of CAM as a model system also allowed discrimination between the observed effects of Friend erythroleukemia cells and of PGE1 on angiogenesis vs vasculogenesis. Thus, observation of the growing embryo allowed the determination of whether the different substances tested affected vasculogenesis, compromising its viability, or selectively affected angiogenesis. MATERIALS AND METHODS Friend erythroleukemia cells (ATCC, Manassas, VA, USA) were routinely maintained in culture medium (Cataldi et al., 2003); briefly, RPMI-1640 medium (Gibco, Milan, Italy) was supplemented with 10% Fetal Calf Serum (FCS) (Gibco, Milan, Italy) and 1% penicillin (10,000 U/ ml)/streptomycin (l0 mg/ml) (Gibco, Milan, Italy), at 37°C in a controlled atmosphere with 5% CO2. HUVEC (ATCC, Manassas, VA, USA) were routinely maintained in complete culture medium containing: endothelial basal growth medium (EBM-2) with 2% fetal bovine serum (FBS), 0.4% human FGF-B, 0.1% VEGF, 0.1% recombinant long R Insulin-like Growth Factor-B (R3IGF-1), 0.1% human Epidermal Growth Factor (h-EGF), 0.1% gentamicin sulfate (GA-1000), 0.04% hydrocortisone, 0.1% ascorbic acid, and 0.1% heparin. All these culture medium components were from Cambrex Bio Science, Milan, Italy. HUVEC were seeded in cell-culture flasks coated with 0.2% gelatin (Sigma Chemical Co., Milan, Italy), at 37°C in a controlled atmosphere with 5% CO2. 3x107 Friend erythroleukemia cells were placed in 75 cm2 culture flasks without FCS and incubated for 48 h. At the end of incubation, conditioned medium (CM) was collected, sequentially centrifuged at 3,000 and 15,000 g for 10 min, respectively, and filtered through 0.22 μm pore-size filters (Ribatti et al., 1998).