Figure 4.

Mechanism of E-cadherin gene induction by 1α,25(OH)₂D₃. (A) Northern blot analysis of E-cadherin mRNA expression in SW480-ADH cells untreated or treated with 10⁻⁷ M 1α,25(OH)₂D₃ for 4 h. Where indicated, cells were pretreated with actinomycin D (Act D, 2 μg/ml) or cycloheximide (CHX, 8 μg/ml) 30 min before 1α,25(OH)₂D₃ addition. 10 μg of poly(A)⁺ RNA was loaded per lane. GAPDH was used as an internal control. (B) Activation of the human E-cadherin gene promoter by 1α,25(OH)₂D₃. SW480-ADH cells were transfected with either −987-TK-Luc plasmid, which contains the genomic sequence from +92 to −987 bp, or −178-TK-Luc containing the sequence from +92 to −178 bp of the human E-cadherin gene. The empty TK-Luc vector was used as control. Transfections were performed as described in Materials and methods. White bars, untreated cells; black bars, cells treated with 10⁻⁷ M 1α,25(OH)₂D₃ during 48 h after transfection. Mean values corresponding to five independent experiments done in triplicate are shown. (C) Lack of effect of 1α,25(OH)₂D₃ on E-cadherin mRNA stability. SW480-ADH cells were pretreated or not pretreated for 30 min with actinomycin D (2 μg/ml) and then incubated in the presence (•) or absence (○) of 1α,25(OH)₂D₃ (10⁻⁷ M), during the indicated times. Northern blot analysis of E-cadherin and GAPDH mRNA expression. Conditions were as above. Two independent experiments gave the same result.