INTERLEUKIN-9 IN HUMAN MYELOID-LEUKEMIA CELLS

Here we review our recent data addressing the role of recombinant huma n (rh interleukin 9 (IL-9) in acute myeloblastic leukemia (AML). We fi rst evaluated the proliferative response of 3 leukemic cell lines and 32 primary samples from AML patients to lL-9 alone and combined with r h-IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF, c-kit ligand). The colony forming ability of le ukemic cells was assessed by a clonogenic assay in methylcellulose, wh ereas the cell cycle characteristics of the same samples were determin ed by the acridine-orange (AO) flow cytometric technique and the bromo deoxyuridine (BRDU) incorporation assay. In addition, the terminal deo xynucleotidyl transferase Assay (TDTA) and standard analysis of DNA cl eavage by gel electrophoresis were used to evaluate induction or preve ntion of apoptosis by IL-9. IL-9, used as a single cytokine, at variou s concentrations stimulated the colony formation of the 3 myeloid cell lines under serum-containing and serum-free conditions end this effec t was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resu lted in the increase of the blast colony formation in all the cases st udied and was the most effective CSF for promoting leukemic cell growt h among those tested in this study including SCF, IL-3, and GM-CSF. Th e addition of SCF to IL-9 demonstrated an additive or synergistic effe ct of the 2 cytokines in 5 out of 8 AML cases tested for their CFU-L g rowth (187 +/- 79 colonies in comparison with 107 +/- 32 CFU-L; p = 0. 05). Positive interaction was also observed when IL-9 aias combined wi th IL-3 and GM-CSF. Studies of cell cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leu kemic cells in S-phase in the majority of the cases evaluated. IL-9 an d SCF in combination resulted in a remarkable decrease of the GO cell fraction (38.2 +/- 24% compared to 58.6 +/- 22% of control cultures; p < 0.05) and induced an increase of G1 and S-phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. Furthermore , in this study, reverse transcriptase-polymerase chain reaction ampli fication (RT-PCR) did not show the constitutive expression of IL-9 mRN A in the cell lines and the AML samples studied at diagnosis. In summa ry, IL-9 may play a role in the development of acute myeloid leukemia by stimulating the proliferation of leukemic cells perhaps through a p aracrine growth loop.