A cDNA for the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells.

Abstract

We have previously demonstrated an estradiol-regulated 24 kDa (24K) protein in human breast cancer tissue culture cells and human tumor biopsies. The presence of 24K correlates well with the presence of steroid hormone receptors. In order to further study the hormonal regulation of the 24K protein and gene, we have isolated cDNA clones corresponding to the 24K mRNA. Poly(A)+ RNA isolated from the MCF-7 human breast cancer cell line was translated in a cell-free translation system containing [35S]-methionine. The translation products were immunoprecipitated with a 24K monoclonal antibody, and the in vitro synthesis of 24K protein was confirmed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. The same poly(A)+ RNA was used to construct an oligo(dT)-primed cDNA library in the lambda gt11 expression vector system. The library was screened with a highly specific polyclonal antibody raised against 24K protein purified by immunoaffinity chromatography. Four recombinant clones reacting with the antibody by virtue of antigen expression were isolated and three were used in hybridization-selected translation. Three clones were able to hybridize specifically to a messenger RNA (mRNA) that yielded a Mr 24,000 protein when translated in vitro and analyzed by SDS/polyacrylamide gel electrophoresis. This protein was also immunoprecipitable by the 24K monoclonal antibody. MCF-7 mRNA size fractionated by formaldehyde-agarose gel electrophoresis was transferred to nitrocellulose paper and hybridized to a nick-translated 24K cDNA clone. A single band of hybridization corresponding to a mRNA size of approximately 0.9-1.0 kilobase (kb) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)