Fig. 6.

Nicotine and NNK treatments enhance mutational susceptibility and cell transformation. Human lung and bladder epithelial cells (BEAS-2B and UROtsa) were treated with NNK (0.5 mM) and nicotine (25 mM for BEAS-2B cells, and 5 mM for UROtsa cells) for 1 h at 37 °C; these treatments render 50% cell killing. (A) UVC-irradiated (1,500 J/m²) or H₂O₂ modified (100 mM, 1 h at 37 °C) plasmid DNAs containing the supF gene were transfected into these cells, and the mutations in control, and nicotine- and NNK- treated cells were detected and quantified as previously described (13, 28). (B) Detection of anchorage-independent soft-agar growth. A total of 5,000 treated cells were seeded in a soft-agar plate. The method for anchorage-independent soft-agar growth is the same as previously described (28). Typical soft-agar growth plates stained with crystal violet were shown. (C) Quantifications of percent of control, nicotine, and NNK-treated cells formed colonies in soft-agar plates.