Figure 3

Influence of H-Ras^{val 12} ovexpression on the growth of T24 cells responding to antisense oligonucleotide and genistein treatments. (A) T24 cells were treated with 5 μ M of the indicated phosphorothioate ODN alone or the ODN and 50 μ M genistein, followed by cell extract isolation and Western blotting with the anti-H-Ras antibody. The intensities of H-Ras^{val 12} protein were quantified with a densitometer, and the Ratio line revealed the percentage of the protein produced (after normalising with the levels of actin protein) in the ODN or/and genistein-treated cells vs untreated T24 cells. The result revealed that the expression of H-Ras^{val 12} was inhibited to a greater degree by treating with the antisense ODN than with the control ODN or genistein, and the effect was strikingly obvious after 72 h incubation. Under all experimental conditions, actin remained relatively unchanged. The control ODN was a 17-mer targeted to human immunodeficiency virus, which was used in parallel with anti-H-ras^{val 12} ODN, as described previously (Chen et al, 1996). (B) With increasing amounts of different ODNs, the growth rates of T24 cell line were determined in the presence or the absence of 50 μ M genistein. The plot reveals that only when the anti-H-ras, but not control, ODN (5 μ M) is added, the growth of T24 cells is inhibited in the absence of genistein and the growth rate is further reduced to below 40% of the untreated cells when the drug is added, suggesting that the expression of H-Ras renders drug-resistant phenotype. The symbols used are: cells without any treatment, □; and cells treated with 0.5% DMSO solvent, ▪; 50 μ M genistein, ; the control ODN, ▨; the control ODN supplemented with 50 μ M genistein, ; the anti-H-ras antisense ODN, ⍁; and the antisense ODN plus genistein, .