Identification of estrogen-responsive genes in neuroblastoma SK-ER3 cells

Abstract

To evaluate the role of estrogen receptor in the differentiation of cells of neural origin, we developed a molecular approach aimed at the identification of estrogen target genes by mRNA differential display PCR (ddPCR) in human neuroblastoma SK-ER3 cells. More than 3000 RNAs were examined, a few of which displayed a differential regulation pattern in response to 17beta-estradiol (E2). Sequence analysis of three differentially amplified ddPCR products showed homology with the growth-associated nuclear protein prothymosin-alpha (PTMA), the Bcl2-interacting protein Nip2, and one mRNA previously described by others in fetal human brain. Two ddPCR products, referred to as P4 and P10, corresponded to new DNA sequences. Northern analysis confirmed that estrogen treatment of SK-ER3 cells resulted in the upregulation and downregulation of expression of these messages. In particular, PTMA was found to accumulate at both 1 and 17 hr after E2 treatment, whereas P10 product accumulated only at 1 hr. Conversely, P4, Nip2, and the fetal brain-related mRNAs were significantly decreased by the treatment. Further time course analysis of PTMA and Nip2 mRNAs levels indicated that the hormone exerted a marked biphasic regulatory effect on expression of both messages during the course of cell differentiation. In the present study we report for the first time the identification of a panel of estrogen target genes in neural cells that provide new insights in the molecular mechanism of action of E2 in cells of neural origin.

Fig. 1.

Fingerprints of RNAs extracted from SK-ER3 neuroblastoma cells and screened by differential display PCR. Total RNAs from control (C) and E₂-treated (1h and 17h) cells were reverse-transcribed in duplicate reactions using a 1 b anchor oligo-dT primer (dT₁₁-G). PCR reactions were performed in duplicate on each cDNA mix using various combinations of dT₁₁-G and arbitrary forward primers (AP-10, AP-11, and AP-12) in the presence of 10 μCi of α-[³⁵S]dATP. The amplified products were then separated on a 6% acrylamide–urea gel and visualized by autoradiography. Arrows point to differentially amplified products in control versus E₂-treated cells identified within one set of ddPCR reactions.

Fig. 2.

Northern analysis of the expression of SK-ER3 messages identified by differential display PCR. Northern blot analyses were performed using digoxigenin-labeled ddPCR products as hybridization probes. Gels were loaded with 10 μg of total RNA from either control (C) or cells treated with E₂ for 1 or 17 hr (1h and 17h). Left panels,Autoradiographs from Northern analysis. Center panels,Ethidium bromide-stained 18 and 28 S rRNAs. Right panels,The relative band intensities were estimated by densitometric analysis of autoradiographs after normalization with respect to 18 and 28 S RNAs. Bars represent the averages of two to five separate determinations. O.D., Optical density.

Fig. 3.

Time course of the effect of E₂ on Nip2 expression in SK-ER3 neuroblastoma cells. Cells were plated in six-well dishes and then grown in the absence or presence of 1 nm E₂ described in Materials and Methods. Total RNAs were extracted at the indicated times and analyzed by Northern blot using a digoxigenin-labeled P6 ddPCR product as the hybridization probe. The relative band intensities (top, a representative experiment) were analyzed by densitometric analysis after normalization to ethidium bromide-stained 18 and 28 S rRNAs. Columnsrepresent means ± SEM (error bars) of six separate determinations. Statistical significance by two-way ANOVA with Tukey multiple range test: p ≤ 0.05 versus control values (1 h and 19 h); p ≤ 0.01 versus control values (2 d and 4 d).O.D., Optical density.

Fig. 4.

Time course of the effect of E₂ on PTMA expression in SK-ER3 neuroblastoma cells. Cells were plated in six-well dishes and then grown in the absence or presence of 1 nm E₂ as described in Materials and Methods. Total RNAs were extracted at the indicated times and analyzed by Northern blot using a digoxigenin-labeled P3 ddPCR product as the hybridization probe. The relative band intensities (top, a representative experiment) were analyzed by densitometric analysis after normalization to ethidium bromide-stained 18 and 28 S rRNAs.Columns represent the means ± SEM (error bars) of six separate determinations. Statistical significance by two-way ANOVA with Tukey multiple range test: p ≤ 0.05 versus control values (17 h); p ≤ 0.01 versus control values (4 d and 6 d). O.D., Optical density.