Abstract
Wild-type as well as variant oestrogen receptor (ER) mRNAs with exon 5 and 7 deleted were identified in a panel of human breast tumour cell lines by reverse transcriptase-polymerase chain reaction followed by dideoxynucleotide sequence analysis, and then quantitated by ribonuclease protection analysis. All cell lines categorised as ER+ by ligand-binding analysis expressed both wild-type and variant ER transcripts. Most cell lines classified as ER- did not express any ER transcript. However, three ER- cell lines (BT-20, MDA-MB-330 and T47Dco) expressed both wild-type and variant transcripts. A differential pattern of expression of wild type to variant was seen in both ER+ and ER- cell lines, however this pattern was not paralleled by differences in ligand-binding activity. Breast tumour cell lines previously classified as ER- expressed significantly lower levels of ER transcripts than did their ER+ counterparts. In view of these findings, as well as earlier reports that the exon 5 deletion ER variant encodes a dominant-positive receptor, it seems clear that some cell lines are misclassified as ER-, and express both wild-type and variant ER mRNAs, and that the overexpression of this variant may account, in part, for their oestrogen-independent phenotype.
Highlights
S_unmary Wild-type as well as vanrant oestrogen receptor (ER) mRNAs with exon 5 and 7 deleted were identified in a panel of human breast tumour cell lines by reverse transcriptase-polymerase chain reaction followed by dideoxynucleotide sequence analysis, and quantitated by ribonuclease protection analysis
We have recently identified variant ER mRNA transcripts containing precise deletions of exon 3, 5 or 7 in breast tumours classified as ER-negative progesterone receptor (PgR) positive or ER positive/PgR negative by ligandbinding analysis (Fuqua et al, 1991, 1992a; McGuire et al, 1992)
We report here that variant ER transcripts with exons 5 and 7 deleted are present in a number of human breast tumour cell lines which had previously been classified as ER positive or ER negative by ligand-binding analysis
Summary
The BT-20, BT-474, MDA-MB-134, MDA-MB-361, MDAMB-435S and MDA-MB-453 cell lines were obatined from the American Type Culture Collection, Rockville, MD, USA. Reverse transcriptase-polymerase chain refrom MCF-7, MDA-MB-361, BT-20 and MDA-MB-330 cells confirmed sequences established for the human ER (Ponglikitmongkol et al, 1988), while the smaller variant fragment was found to contain wild-type sequences for exons 4 and 6 action products were gel purified and cloned into pGEM7zf(+) vectors (Promega, Madison, WI, USA). Reverse transcriptase-polymerase chain reaction amplification of total RNA from the four cell lines mentioned above was performed using the primers HB18 and ERY6 to generate cDNA fragments from nucleotides 1553-2042 of the ER mRNA (Figure lb). Sequence analysis of the cloned 305 bp product from MDAMB-330 (Figure 2) as well as other cell lines (MCF-7, BT-20 and MDA-MB-361) revealed wild-type sequences for exons 6 and 8 with exon 7 deleted. The MDAMB-231 cell line failed to express any ER mRNA transcripts
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