A functional assay in yeast for the human estrogen receptor displays wild-type and variant estrogen receptor messenger RNAs present in breast carcinoma.
Human estrogen receptor (hER) variants or mutants with altered functional activity may be responsible for resistance to the antiestrogen tamoxifen in breast cancer. The method presented in this report is a screening method for functional activity of hER in yeast Saccharomyces cerevisiae. hER mRNA isolated from breast cancer tissue is subjected to reverse transcription-PCR, directly cloned into a yeast expression vector in vivo, and subsequently tested for functional activity in a simple yeast growth assay. This technique, functional analysis of separated alleles in yeast of the human estrogen receptor (hER-FASAY), gives a display of the prevalence and functional activity of all of the variant hER mRNAs among normal, wild-type receptors in a breast tumor sample. The hER-FASAY can discriminate among wild-type hER, constitutively active hER, and inactive hER. In contrast to standard immunohistochemistry, this assay gives insight into the functional activity of hER. The hER-FASAY was optimized and validated using breast cancer cell lines MCF-7 and T47D and seven breast cancer biopsies. Phenotypes detected with the hER-FASAY were validated by DNA sequencing. In both cell lines and tumor biopsies, hER variants are highly common and mainly caused by alternative RNA splicing, whereas point mutations and deletions occur only at low frequency.
van Dijk
M A
MA
Department of Pathology, The Netherlands Cancer Institute, Amsterdam.
Floore
A N
AN
Kloppenborg
K I
KI
van 't Veer
L J
LJ
D001681:Biological Assay / Q000379:methods*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis* / Q000235:genetics; D011980:Receptors, Progesterone / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone