Altered expression of myeloid markers by modulation of an isoprenyl pathway

Proc Amer Assoc Cancer Res, Volume 47, 2006 3826 The cause of myeloproliferative diseases polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofiborosis (IM) is unknown although recent work suggests JAK-STAT signaling is highly relevant. However, alterations in these pathways do not provide a complete explanation for the initiation and establishment of hyper-proliferation states, and it is important to investigate potential mechanisms responsible for the progression of these disorders. We used clusters of cell markers as tools to determine which pathways may be altered in myeloproliferation. The well established and defined HL60 cell line was used as a model system as it can be differentiated toward monocyte or granulocyte (PMN) lineages. We were particularly interested in the induction of CD177 or PRV1 since it is overexpressed in 95% of PV patients and the pathways responsible for its expression are unknown. HL60 cells were analyzed by flow cytometry and modified cell staining. The markers analyzed included CD11b (C3bi receptor present on PMNs, monocytes and NK cells), CD15 (Lex, present on mature PMNs and monocytes), CD16 (FcγRIII, present on mature PMNs), CD18 (cell adhesion, present on leukocytes), and CD177 (Ly6 superfamily protein of unknown function, possible early marker of proliferation, upregulated in PV and some ET and IM patients). Annexin V (apoptosis) levels were also analyzed. The HL60 cells were exposed to IGF-1, prolactin, PMA, DMSO, ATRA, B581 (farnesyl transferase inhibitor), trans,trans -farnesol (Farnesol), and physical stress (heat shock at 42oC, hypotonic and hypertonic osmotic stress). IGF-1, hypotonic shock and Farnesol induced CD177 to varying degrees. Only Farnesol treatment resulted in significant changes to a granulocyte differentiation type with concurrent 50% cell loss due to apoptosis. PMA induced monocyte differentiation as expected with an increase in monocytic marker expression (CD11b, CD15, and CD18). Hypertonic and brief heat stress did not result in marker changes, expect for CD177 expression which was somewhat inhibited by hypertonic stress. Although the farnesyl transferase inhibitor did not affect marker expression, Farnesol had a powerful effect on CD177, and resulted in a greater than 50% differentiation toward granulocyte lineage. It was also synergistic with hypotonic stress, IGF-1 treatment, and DMSO treatment. IGF-1 effects are modulated through JAK-STAT pathways suggesting a role for these in CD177 expression. However, Farnesol had a much more potent effect on marker expression, and its effects were maximized within 24 hours of administration. Farnesol may modulate isoprenylation dependent pathways including key targets of the ras oncogene protein. We conclude that cellular proliferation, differentiation and CD marker gene expression can be highly altered by Farnesol and modulation of isoprenylation pathways and these pathways may be important in myeloproliferative diseases.