Figure 3

Transfection of human DF with anti-α3 antisense oligonucleotides (AsOs) abolishes Nic-induced changes in cell cycle markers. A, Intracellular accumulation of FITC-labeled α3 AsOs. FITC-labeled AsOs (0.5 μm; Table 4) was added to second passage human foreskin DF. Localized FITC-labeled AsOs was viewed live via phase-contrast fluorescence microscopy after a 24-hour incubation; magnification × 400. Note: anti-α3 AsOs is distributed into the nucleus as well as the cytoplasm. Control oligonucleotide was similarly distributed (not shown). B, Effect of anti-α3 AsOs on the α3 nicotinic acetylcholine receptor (nAChR) subunit protein in human DF. The cells were seeded in 24-well plates at a density of 5 × 10⁴/well and incubated in a 5% CO₂ incubator for 72 hours in fibroblast growth medium (FGM) in the presence of LipofectAMINE PLUS alone (control), 20 nm sense oligonucleotide, or 20 nm each of three phosphorothioated AsOs (Table 4). The monolayers were washed, the cells were scraped, and the cellular proteins were analyzed by WB as detailed in “Materials and Methods.” In each lane, 20 μg of total protein was subjected to electrophoresis, blotted, and probed with a rabbit anti-α3 Ab characterized previously (Nguyen et al, 2000). The α3 band appeared at the expected molecular weight of 60 kDa. The anti-α3 AsOs dramatically reduced the intensity of the 60-kDa receptor band. Densitometric analysis showed at least 90% reduction of α3, compared with control. Control (ie, sense) oligonucleotide only moderately altered the amount of α3 protein. The ratio data are the means ± sd of the values obtained in at least three independent experiments. The images represent typical appearance of the bands in gels. The staining of the receptor bands was absent in the negative control experiments in which the membranes were treated without primary Ab, with irrelevant primary Ab of the same isotype and host, or the antipeptide antisera before treatment of the blotting membrane was preincubated with the α3 peptide used for immunization (not shown). Asterisk indicates significant (p < 0.05) differences from control. C, Changes in the expression of cell cycle markers in AsOs-transfected DF treated with Nic. Relative amounts of p21, PCNA, cyclin-D1, and Ki-67 were analyzed by WB of the total protein isolated from human DF transfected with anti-α3 AsOs or with sense oligonucleotide or from nontransfected DF (Control) after 24-hour incubation in FGM containing no drugs or 10 μm Nic with or without 50 μm Mec. The cell cycle marker protein contents were measured by standard densitometry of the specific bands in the gels, as described in “Materials and Methods.” The gene expression ratio of 1 was given to the drug-untreated, control DF. The ratio data are the means ± sd of the values obtained in at least three independent experiments. The images represent typical appearance of the bands in gels. A dramatic decrease of the relative amounts of individual cell cycle markers was found in the drug-untreated DF transfected with anti-α3 AsOs. The transfection with anti-α3 AsOs also abolished Nic-induced changes in the cell cycle markers, which could be observed in the intact DF and DF transfected with sense oligonucleotide. In the latter cultures, Mec abolished Nic effects. The staining of the protein bands was absent in the negative control experiments in which the membranes were treated without primary Ab or with irrelevant primary Ab of the same isotype and host (not shown). Asterisks indicate significant (p < 0.05) differences from control.