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 2Delta, exon 3Delta, exons 2-3Delta, exon 4Delta, exon 5Delta, exon 6Delta and exon 7Delta. 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 2Delta 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 3Delta specific primer amplified two transcripts that have deletions in exon 3, and exons 3 and 7. The exon 2-3Delta 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 4Delta specific primer amplified two products that have deletions in exon 4, and exons 4 and 7. The exon 5Delta specific primer amplified three transcripts, that have deletions in exon 5, exons 5 and 2, and exons 5, and 2-3. The 6Delta specific primer amplified only one transcript that has a deletion in exon 6. The 7Delta 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 2Delta and exon 4Delta transcripts. Analyses in the tumor samples indicated that the above transcripts are extensively modified.