Isolation of Estrogen Regulated Genes from MCF-7 Human Mammary Cancer Cells
Abstract
The established human mammary epithelial cell line MCF-7 has been extensively employed as an in vitro model system for the study of hormonally responsive human breast cancer. These cells possess specific receptors for estradiol (E2) and physiological concentrations of E2 are required to support cell growth both in vitro and in vivo. However, although E2 alters steady-state levels of specific mRNA transcripts, no cloned estrogen-responsive genes are known to regulate MCF-7 cell growth. This investigation has focused on the isolation of estrogen-regulated cDNA clones from cell cultures under conditions that maximize the stimulatory effect of E2 on cell growth. MCF-7 cells were maintained under conditions in which cells were either quiescent (estrogen-deprived, E-), or rapidly dividing (estrogen-treated, E+). After seven days the cell number of the E+ cultures increased 3-4-fold while the number of E- cells remained constant. Additionally pulse-labeled E+ cultures incorporated up to 30-fold more [3H] thymidine than E- cultures. E2 also altered the mRNA expression of three previously isolated cDNAs: pS2 (induced 30-100-fold), 36B4 (unaffected), and gelsolin (decreased 10-fold). R0t and C0t analyses confirmed that an E+ cDNA library later constructed in lambda gt10 accurately reflected the parent RNA population. The primary library was then screened using a E+ radiolabeled single-stranded cDNA selected by hybridization against a 15-fold excess of E- poly(A) RNA. Northern blots confirmed that all of the final four distinct selected clones (pMT1, pMT2, pMT3, pMT4) were induced by estrogen treatment. pMT1 was subsequently identified as progesterone receptor, a known estrogen-regulated gene. pMT2 was subcloned into pGEM3Z as two separate EcoRI fragments of 2.2 and 4.5 kb. The cDNA hybridized to a single mRNA species of approximately 11 kb and the sequence of this clone did not correspond to any gene registered in GENBANK.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 13, 1990
- Accession Number
- AD1010950
Entities
People
- Susan Aitken
Organizations
- Uniformed Services University of the Health Sciences