Abstract
The glutathione S-transferase pi gene (GST pi) is highly expressed in estrogen receptor negative (ER-) but not expressed in ER+ human breast cancer cell lines. To define regulatory mechanisms of GST pi gene expression, we analyzed both the activity of the GST pi promoter and the posttranscriptional fate of GST pi RNA sequences in three ER+ and three ER- breast cancer cell lines. Expression of a transiently transfected CAT reporter gene driven by the GST pi promoter and 2203 nucleotides of 5'-flanking sequences were similar in all six cell lines regardless of ER status. Endogenous GST pi transcription rates in nuclei isolated from ER- cells were quite low despite high steady state levels of cytoplasmic mRNA. Furthermore, the endogenous GST pi gene was transcribed in ER+ nuclei at rates similar to those obtained in ER- nuclei. We determined the stabilities of mRNAs transcribed from the endogenous GST pi gene (ER- cells) and from a stably transfected GST pi cDNA expression vector (ER+ and ER- cells). The endogenous GST pi mRNA was extraordinarily stable in ER- cells. Comparisons between transfected ER+ and ER- cells revealed no significant differences in the stabilities of transfection-derived GST pi mRNA sequences. We conclude that GST pi mRNA stability contributes significantly to the high levels of cytoplasmic mRNA observed in ER- cells, but that the differential expression of GST pi in ER+ versus ER- cells is governed by other posttranscriptional processes.
Highlights
But not expressed in ER+ human breast cancer cell lines
Cell Culture and DNA Transfection-Three ER+(WT MCF7, ZR75B, and T47D) andthree ER- (ADRrMCF7, HS578T, and MDA231) human breast cancer cell lines were grown in improved minimum Eagle'smedium (GIBCO) supplemented with 10% fetal calf serum at 37 "C in 5% COZ.Transfection of expression vector DNA was accomplished by the calcium phosphate-DNA precipitate method described [25]
GSTa mRNA Stabilities-The relative stabilities of endoginvestigate these possibilities, we examined the stabilities of endogenous GSTa mRNA (ER- cells) and of GSTa mRNA sequences derived from the transcription of a stably trans
Summary
Cell Culture and DNA Transfection-Three ER+(WT MCF7, ZR75B, and T47D) andthree ER- (ADRrMCF7, HS578T, and MDA231) human breast cancer cell lines were grown in improved minimum Eagle'smedium (GIBCO) supplemented with 10% fetal calf serum at 37 "C in 5% COZ.Transfection of expression vector DNA was accomplished by the calcium phosphate-DNA precipitate method described [25]. 16 pg of a GSTx cDNA expression vector under the controI of a CMV promoter (pHDG)(6) was co-transfected with 4 pgofpSVNeo. Transgenic colonies were selected in G418 (GIBCO) at drug concentrations predetermined to be sufficient to completely inhibit the growth of nontransfected cells. Transgenic colonies were selected in G418 (GIBCO) at drug concentrations predetermined to be sufficient to completely inhibit the growth of nontransfected cells These G418 concentrations were 0.3 mg/ml for ADRrMCF7 cells, 0.75 mg/ml for ZR75B and T47D cells, 1 mg/ml for WT MCF7 and HS578T cells, and 2 mg/ml for MDA231 cells. RNAs were prepared from cells harvested at various times following actinomycin D treatment, and the levels of GSTx mRNA sequences were quantitatively determined by RNase protection (above) or by slot blot analysis. Resistant colonies were screened for the presence of transfection vector-derived GSTx gene DNA and mRNA (below).Additional WT
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