Figure 2.

Effects of zinc stimulation on expression level and silencing of β-geo in K562 cell clones. (A) The basal and induced expression of β-geo in eight clones carrying single integrated copies of MT/β-geo; bars denote SEM. Results shown are means of at least three experiments, and are normalized to the negative control. There is a 5-fold variation of basal expression level between clones (open bars). Induced levels (solid bars) are 24 h after addition of 80 μM zinc sulfate. The basal and induced levels of β-gal in cells maintained free of G418 do not differ significantly from those shown, except in cases in which silenced cells exist in the assayed population; in those cases the levels are proportional to the percentage of expressing cells. (B) Silencing of β-geo expression in MT/β-geo clones is retarded by zinc stimulation. Cells were continuously expanded with (+Zn) or without (−Zn) 80 μM zinc sulfate in the culture medium. The x axis in these histograms represents a three-decade log scale of fluorescence, and the y axis represents the relative cell number. All clones were maintained in G418 until time 0 and then split into induced and uninduced aliquots, so that at time 0 all were expressing and the two populations were identical. There is a steady accumulation of silenced cells over time. Weeks and years are denoted as wk and yr, respectively. The histograms show progressive silencing over a period of up to 1 yr. Two clones (C2 and C13) show no silencing. The other clones show significant, but varying, proportions of silent cells, but in each case zinc stimulation slows or completely prevents silencing. In the case of clone 10, silencing in both aliquots was complete by 33 wk so that later time points are not shown. The uninduced (−Zn) aliquots of clones 14 and 20 were lost after 33 wk and so there is no 1-yr time point.