Three per cent of nsc 34 cells trans fected with

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cell death. Three per cent of NSC-34 cells trans- fected with control siRNA showed fragmented DNA at 48 h, as indicated by a horseradish-peroxi- dase positive dark stain under microscopic inspec- tion (FIG. 3). Transfection with SMN siRNA sig- nificantly increased the percentage of TUNEL- positive NSC-34 cells to 9.8% of counted cells ( p <0.05 vs control). At 72 h after transfection with control siRNA the percentage of TUNEL-positive cells was not significantly increased ( p =0.188), while 37.4% of SMN siRNA transfected NSC showed DNA fragmentation ( p <0.0001 vs control). Mean and range of number of cells counted were: Control 48 h: 134 [32-260]; SMN 48 h 269 [36-440]; Control 72 h 147 [58-240]; SMN 72 h 258 [138-440]. These data confirm that lowered smn levels resulted in apoptotic cell death of NSC-34 cells. NSC-34 Cell Viability Following SMN Adenoviral Overexpression The next experiments investigated whether smn has a protective effect against apoptosis and cell death. Staurosporine was used to evoke apoptosis in NSC-34 cells and smn production induced using adenoviral gene transfer. Relative cell viability by MTT test was signifi- cantly decreased by 500 nM staurosporine to 57% of the control culture ( p <0.0001, FIG. 4A). In con- trast, AdV-SMN treated NSC-34 cells were at 94% of control culture viability after staurosporine expo- sure, showing significantly less vulnerability to staurosporine toxicity than the untreated or control ADV-LacZ treated cells (both p <0.0001). Overexpression of smn by AdV in NSC-34 cells was associated with a decrease in caspase-3 activity induced by staurosporine (FIG. 4B). NSC-34 cells exposed to staurosporine alone (500 nM final con- centration) for 12 h produced a high level of cas- pase-3 activity similar to the control DNase treat- ment ( p =0.892). However, NSC-34 cells treated with AdV-SMN showed significantly less caspase-3 activity compared to both those treated with DNase or staurosporine (both p <0.0001). Horseradish-peroxidase positive dark stain was observable in 4% of control NSC-34 cells (22 out of 558 counted cells) observed at 96 h after plating. At the same time point, almost 12% (80 / 654) of counted cells exposed to staurosporine (500 nM) showed DNA fragmentation. This was reduced to ~7% of cells treated with AdV-SMN (45/604). DISCUSSION Spinal muscular atrophy is caused by the selective loss of anterior horn cells, which subsequently leads to a muscle weakness and atrophy. Mutations in the telomeric SMN gene result in a low protein product, which is intolerable for lower motor neu- rons. In this study, our goal was to test whether regulation of smn affected apoptotic cell death in an in vitro motor neuron model. The same target sequence in the SMN gene was selected that was shown to be used successfully by others in vitro (Trulzsch et al. , 2004). Suppression of smn produc- tion by RNA interference resulted in a progressive decrease in smn protein to almost 60% of untreated control three days following transfection. These results showed that the in vitro neuronal system achieves levels of smn similar to that observed in human SMA cells and cells from transgenic SMA mice (Hsieh-Li et al. , 2000; Monani et al. , 2000b).
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