Figure 6.
Figure 6. Effect of ATO treatment on expression the NF-κB-dependent gene, FLIP. (A) ML1 cells were exposed to ATO and the IκB kinase inhibitor BMS 345541 for 2 hours, and TNF-α was added for 1 hour. RNA was extracted, and FLIP mRNA levels were determined using quantitative real-time PCR. Values for each gene were normalized to expression levels of β2-microglobulin. Message levels for FLIPLong were substantially higher than for FLIPShort, resulting in a high FLIPLong/FLIPShort ratio, similar to that seen in advanced MDS. Inhibition of IKBα kinase by BMS345541 predictably blocked FLIP message. ATO reduced FLIPLong message (as expected, due to NF-κB inhibition), and TNF-α enhanced FLIPShort mRNA above the level of untreated controls. What factors are responsible for differences in the responses of the 2 splice variants is currently not clear. Shown are results achieved with ATO at 5μM, TNF-α at 20 ng/mL, and BMS345541 at 5μM. (B) ML1 cells transduced to overexpress FLIPLong were treated as indicated and evaluated for apoptosis by Annexin V and PI staining, GFP-tranduced cells served as control. Results represent the mean plus or minus SE of 3 independent experiments, each carried out in duplicates. FLIPLong overexpression reduced the rate of apoptosis in response to ATO at all doses tested (*P < .05; paired t test). Values are corrected for background apoptosis as described in “Materials and methods.” (C) ML1 wild-type cells, ML1 cells transduced with GFP control vector (GFP) and with FLIPLong (FLIPLong), respectively, were assessed for FLIPLong expression by Western blot. (D) Spontaneous NF-κB binding as determined by 32P NF-κB gel shift of nuclear extracts from control-transduced (GFP) and FLIPLong-overexpressing (FLIPLong) ML1 cells. (E) GFP and FLIPLong cells were exposed to ATO for 1 hour, followed by TNF-α or TNF-α only and harvested 1 hour later. Phosphorylated IKBα (IKBα-P) was determined by Western blot. Results shown were obtained with ATO at 10 μM and TNF-α at 20 ng/mL. Thus, ATO down-regulated FLIP, but FLIPLong overexpression was associated with increased NF-κB activity and ATO resistance.

Effect of ATO treatment on expression the NF-κB-dependent gene, FLIP. (A) ML1 cells were exposed to ATO and the IκB kinase inhibitor BMS 345541 for 2 hours, and TNF-α was added for 1 hour. RNA was extracted, and FLIP mRNA levels were determined using quantitative real-time PCR. Values for each gene were normalized to expression levels of β2-microglobulin. Message levels for FLIPLong were substantially higher than for FLIPShort, resulting in a high FLIPLong/FLIPShort ratio, similar to that seen in advanced MDS. Inhibition of IKBα kinase by BMS345541 predictably blocked FLIP message. ATO reduced FLIPLong message (as expected, due to NF-κB inhibition), and TNF-α enhanced FLIPShort mRNA above the level of untreated controls. What factors are responsible for differences in the responses of the 2 splice variants is currently not clear. Shown are results achieved with ATO at 5μM, TNF-α at 20 ng/mL, and BMS345541 at 5μM. (B) ML1 cells transduced to overexpress FLIPLong were treated as indicated and evaluated for apoptosis by Annexin V and PI staining, GFP-tranduced cells served as control. Results represent the mean plus or minus SE of 3 independent experiments, each carried out in duplicates. FLIPLong overexpression reduced the rate of apoptosis in response to ATO at all doses tested (*P < .05; paired t test). Values are corrected for background apoptosis as described in “Materials and methods.” (C) ML1 wild-type cells, ML1 cells transduced with GFP control vector (GFP) and with FLIPLong (FLIPLong), respectively, were assessed for FLIPLong expression by Western blot. (D) Spontaneous NF-κB binding as determined by 32P NF-κB gel shift of nuclear extracts from control-transduced (GFP) and FLIPLong-overexpressing (FLIPLong) ML1 cells. (E) GFP and FLIPLong cells were exposed to ATO for 1 hour, followed by TNF-α or TNF-α only and harvested 1 hour later. Phosphorylated IKBα (IKBα-P) was determined by Western blot. Results shown were obtained with ATO at 10 μM and TNF-α at 20 ng/mL. Thus, ATO down-regulated FLIP, but FLIPLong overexpression was associated with increased NF-κB activity and ATO resistance.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal