Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach

Estrogen receptor (ER) alpha splice variant transcript profiles were analyzed by RT PCR in six ER positive breast cancer cell lines, MCF-7, T47D, ZR-75, LCC1, LCC2 and LCC9, three ER negative cell lines, MDA-MB-435, MDA-MB-235 and LCC6, and three ER positive malignant breast tumors using targeted primers which specifically anneal to the splice junctions of exon 2 Delta, exon 3 Delta, exons 2-3 Delta, exon 4 Delta, exon 5 Delta, exon 6 Delta and exon 7 Delta. The partner primers were chosen such that largest possible transcripts were amplified between exons 1 and 8. The results described here show that each splice specific primer amplified not only the single exon deleted transcript but also a number of related transcripts that have deletions in various combinations of exons. The exon 2 Delta specific primer amplified five transcripts that have deletions in exon 2, exons 2 and 7, exons 2, 5, and 7, exons 2;and 4-5, and exons 2 and 4-6. The exon 3 Delta specific primer amplified two transcripts that have deletions in exon 3, and exons 3 and 7. The exon 2-3 Delta specific primer amplified three products that have deletions in exons 2-3, exons 2-3 and 7 and exons 2-3, 5 and 7. The exon 4 Delta specific primer amplified two products that have deletions in exon 4, and exons 4 and 7. The exon 5 Delta specific primer amplified three transcripts, that have deletions in exon 5, exons 5 and 2, and exons 5, and 2-3. The 6 Delta specific primer amplified only one transcript that has a deletion in exon 6. The 7 Delta specific primer amplified four transcripts, that have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 and 3-5. None of the above splice specific primers amplified the wild type ER sequences. The six ER positive cell lines differed in the patterns of the variant transcripts and among the three ER negative cell lines analyzed, only MDA-MB-435 showed the presence of exon 2 Delta and exon 4 Delta transcripts. Analyses in the tumor samples indicated that the above transcripts are extensively modified. (C) 2000 Elsevier Science Ltd. All rights reserved.