Involvement of NO in FTI-induced inhibition of CML cell viability. (A) iNOS mRNA signal was amplified in CML cells cultured for 48 hours in the presence of a representative FTI (SCH [1 μM]). Ethidium bromide staining of RT-PCR products performed with specific primers for iNOS and GAPDH. GAPDH documents equal RNA loading. The positive control was supplied by the manufacturer. (B) FTIs caused increased expression of iNOS protein in cells from 2 CML patients and in LAMA and K562 cells (CML1 and K562 treated with Man-A, 50 μM; CML2 treated with SCH [1 μM]). Actin is used as control for constant protein loading. (C) A representative FTI (SCH [1 μM]) caused intracellular NO production in LAMA cells. NO production was measured using the cell-permeable fluorescent indicator DAF-2 DA. γ-MM-arg, a competitive inhibitor of iNOS, partially abrogated FTI-mediated NO production. (D) Inhibition of iNOS partially blocks FTI-induced inhibition of CML cell viability. γ-MM-arg (500 μM), a competitive inhibitor of iNOS, induced a partial reversion of CML cell viability inhibition induced by all FTIs. Bars represent mean ± SEM of 3 experiments. Cumulative mean ± SEM of cell viability after FTI exposure at IC50 and IC10: 54.3% ± 3% and 66.3% ± 3% versus 62.6% ± 3% and 86.6% ± 3% in absence and in presence of γ-MM-arg; P = .056 and P = .01, respectively. (E) γ-MM-arg partially prevented FTI-mediated apoptosis. Flow cytometric detection of apoptotic hypodiploid DNA derived from a representative CML patient treated with SCH (1 μM). Horizontal bars indicate hypodiploid DNA peak and its percentage.