The DR-2 element in the 3′ enhancer of the Epo gene is RARE in P19 cells but not in Hep3B cells.
P19 cells (A) and Hep3B cells (B) show transient expression of Luc reporter constructs. Characteristics of the plasmids are shown in Figure 3 and Table 1. After transfection, the cells were cultured in 21% oxygen for 20 hours in the presence and absence of 5 × 10−7 mol/L all-trans RA. The inducibility by RA (the ratio of Luc activity in the presence versus absence of RA) is shown. The inducibility of pWT construct was defined as 1. Each value is the mean ± SD of triplicate experiments. (C) The 3′ enhancer from the human Epo gene acts as RARE in P19 aggregates. (D) The 3′ enhancer from the human Epo gene responds to hypoxia in Hep3B cells. Permanent P19 and Hep3B cell lines harboring the β-galactosidase reporter construct were established (P19/Z and Hep3B/Z). The reporter gene is under the control of the human Epo promoter (hP) and the 3′ enhancer (hE) (Figure 3B, bottom). (C) Parental P19 cells and P19/Z cells were cultured in aggregates with or without 5 × 10−7 mol/L all-trans RA for 48 hours in 21% oxygen or were cultured for 28 hours in 21% oxygen and then for 20 hours in 2% oxygen. After 48 hours, the cells were stained with X-gal solution. (D) Parental Hep3B cells and Hep3B/Z cells were cultured with or without 5 × 10−7 mol/L all-trans RA for 24 hours in 21% or 2% oxygen. Magnifications are 25-fold.
