Figure 1

Gene expression changes of the cell cycle and apoptosis markers in human dermal fibroblasts (DF) exposed to nicotine (Nic). Total RNA and proteins were isolated from human DF treated for 24 hours with 10 μm Nic in the absence or presence of 50 μm mecamylamine (Mec). The relative amounts of mRNA transcripts and protein levels of the cell cycle markers were measured and the results expressed as described in “Materials and Methods.” Asterisks indicate significant (p < 0.05) differences from control. A, The mRNA levels of the cell cycle genes p21, cyclin D1, PCNA, and Ki-67 and the cell apoptosis markers Bcl-2 and caspase 3 determined by RT-PCR using specific primers (Table 1) and cDNA from exposed versus nonexposed (Control) DF. A PCR product of the expected size was amplified by each primer set specific for p21 (305 bp), cyclin D1 (311 bp), PCNA (320 bp), Ki-67 (452 bp), Bcl-2 (493 bp), and caspase-3 (473 bp). The levels of mRNAs coding for p21, cyclin D1, PCNA, Ki-67, Bcl-2, and caspase 3 were increased in the exposed, compared with control, DF. Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene product was used to normalize the cDNA content in each sample and as a positive control for RT-PCR effectiveness. 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. B, The cell cycle proteins p21, cyclin D1, PCNA, and Ki-67 and the antiapoptotic protein Bcl-2 and the proapoptotic enzyme caspase 3 visualized in Western blots. The molecular weight of each protein is shown in kDa to the right of the gels. In Nic-treated cultures, the relative amounts of the studied cell cycle proteins increased in the range from 1.8- to 2.5-fold, and Mec abolished most of these changes. 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. 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).