Cytokine regulation of inducffile nitric oxide synthase (nos2) and nos2 inhibitor-induced apoptosis and death in chronic lymphocytic leukemia cells
Chronic lymphocytic leukemia (B-CLL) is a malignancy of a mantle zone-based subpopulation of anergic, self-reactive, activated CD5+ B lymphocytes devoted to the production of polyreactive natural autoantibodies. B-CLL is characterized by the accumulation of long-lived non-dividing CD5+ B cells in G0 of the cell cycle. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells is dependent on the autocrine production of NO by NOS2. Inhibition of NOS2 induces BCLL cell apoptosis. The purpose of this study to determine whether cytokine factors that prevent spontaneous in vitro apoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity and NO production and prevent NOS2 inhibitor-induced B-CLL cell apoptosis. Cells were from patients with CD5+ B-CLL with WBC20,000/uL; all had not received leukemia therapy within the last 4 weeks. Peripheral blood mononuclear cells (PBMC) were isolated from blood using ficoll-Hypaque, and T cells and monocytes were depleted using magnetic beads coupled with anti-CD2 and anti-CD 14 antibodies. The resultant cells were 90±2% CD5+/CD19+ and 3±2% CD5-/CD19+ (N=45). We found that B-CLL cells expressed NOS2 as determined by an enzyme assay (8 fold greater expression in BCLL cells than in normal PBMC), by immunoblot (0/12 positive for NOS2 in normal PBMC vs 12/15 positive in CLL), and by RT-PCR analysis (0/10 positive for NOS2 in normal PBMC vs 13/13 positive in B-CLL cells). IL-4 and IFNy significantly increased BCLL cell NOS2 enzyme activity and protein expression during in vitro culture. However. IFNct, nerve growth factor, IL-6, IL-2, IL-8, and G-CSF had no significant effects. We were unable to detect increased concentrations of nitrite or nitrate (surrogate markers of NO production) in B-CLL cell cultures treated with IL-4 or IFNy. IL-4 and IFNy significantly inhibited NOS2 B-CLL cell death and apoptosis induced by the NOS2-specific inhibitor L-N6-(!-iminoethyl)-lysine (L-NIL) or the nonspecific NOS inhibitor NG-monomethylL-arginine (NMMA). In summary, we found that B-CLL cells expressed NOS2, that IL4 and IFNy increased B-CLL NOS2 expression, and that IL-4 and IFNy prevented NOS2 inhibitor-induced B-CLL cell death and apoptosis. Expression of NOS2 by B-CLL cells may promote their survival. NOS2 and NO may represent new molecular targets in the treatment of B-CLL.
Levesque, MC; Misukonis, MA; O'Loughlin, CW; Lee Wilson, D; Adams, DJ; Silber, R; Brice Weinberg, J
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