Individual and combined effects of antisense oligodeoxynucleotides to heavy and light ferritin subunits on the labile iron pool, pro-oxidant-induced reactive oxygen species production, and iron content of cells
| AS added (48-h incubation) . | H-FT* . | L-FT* . | Normalized LIP* . | ROS production* . | Protein oxidation* . | Iron content (μM) . |
|---|---|---|---|---|---|---|
| None | 100 ± 25 | 100 ± 18 | 100 ± 0.36 | 100 ± 46 | 100 ± 4.47 | 727 ± 63 |
| Inversed H-FT | 94 ± 32 | 151 ± 22 | 94 ± 0.54 | 108 ± 0.38 | 112 ± 1.23 | 955 ± 347 |
| H-FT | 24 ± 7† | 270 ± 32‡ | 150 ± 18‡ | 231 ± 0.77† | 99 ± 4.8 | 615 ± 17 |
| L-FT | 169 ± 17‡ | 45 ± 14‡ | 121 ± 9‡ | 192 ± 19‡ | 112 ± 2.3 | 735 ± 22 |
| H- and L-FT | 18 ± 4† | 21 ± 14‡ | 259 ± 2.68† | 270 ± 38‡ | 152 ± 1.9† | 795 ± 30 |
| AS added (48-h incubation) . | H-FT* . | L-FT* . | Normalized LIP* . | ROS production* . | Protein oxidation* . | Iron content (μM) . |
|---|---|---|---|---|---|---|
| None | 100 ± 25 | 100 ± 18 | 100 ± 0.36 | 100 ± 46 | 100 ± 4.47 | 727 ± 63 |
| Inversed H-FT | 94 ± 32 | 151 ± 22 | 94 ± 0.54 | 108 ± 0.38 | 112 ± 1.23 | 955 ± 347 |
| H-FT | 24 ± 7† | 270 ± 32‡ | 150 ± 18‡ | 231 ± 0.77† | 99 ± 4.8 | 615 ± 17 |
| L-FT | 169 ± 17‡ | 45 ± 14‡ | 121 ± 9‡ | 192 ± 19‡ | 112 ± 2.3 | 735 ± 22 |
| H- and L-FT | 18 ± 4† | 21 ± 14‡ | 259 ± 2.68† | 270 ± 38‡ | 152 ± 1.9† | 795 ± 30 |
All AS-ODN concentrations were 1 nM. FT levels were measured by enzyme-linked immunosorbent assay, described in “Materials and methods.” Levels of FT in the untreated cells were 308 ± 78 and 76 ± 14 ng/mg protein for H-FT and L-FT, respectively. Those control levels varied among experiments within the ranges of 150 to 400 and 50 to 150 ng/mg protein for H-FT and L-FT, respectively. DFO treatment (50 μM for 24 hours) was used as a positive control for repression of expression of both FT subunits.24 LIP levels were measured by the calcein-based method2 in cells taken from exponentially growing cultures, and their values were normalized to intracellular calcein. LIP level of the untreated cells here was 1.12 ± 0.004 μM and varied among experiments within the range of 0.08 to 0.14. ROS production was determined 100 seconds after 5 μM H2O2 challenge by the CDCF method, as described in “Materials and methods.” Its level in the untreated cells here was 2.6 ± 1.20 nM CDCF oxidized/second and varied among experiments within the range of 1 to 3 nM CDCF oxidized/second. Protein oxidation was measured by the carbonyl assay,22 as described in “Materials and methods.” Its level in the untreated cells was 10.2 ± 0.45 nmol CO/mg protein and varied among experiments within the range of 10 to 20 nmol CO/mg protein. Ferric ammonium citrate treatment (50 μM for 48 hours) resulted in levels of protein oxidation similar to those attained by the combined AS treatment against hours and L-FT.
AS-ODN indicates antisense oligodeoxynucleotides; H-FT and L-FT, heavy and light ferritin subunits; LIP, labile iron pool; ROS, reactive oxygen species.
Values are in percentage of untreated cells. Values for the AS-ODN–treated cells were different from the values in the untreated cells. Values were determined by independent t test. Data are from 1 of 3 representative experiments, each performed in triplicate.
P = .01.
P = .05.