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 analyz ed by RT PCR in six ER positive breast cancer cell lines, MCF-7, T47D, ZR-7 5, LCC1, LCC2 and LCC9, three ER negative cell lines, MDA-MB-435, MDA-MB-23 5 and LCC6, and three ER positive malignant breast tumors using targeted pr imers which specifically anneal to the splice junctions of exon 2 Delta, ex on 3 Delta, exons 2-3 Delta, exon 4 Delta, exon 5 Delta, exon 6 Delta and e xon 7 Delta. The partner primers were chosen such that largest possible tra nscripts were amplified between exons 1 and 8. The results described here s how that each splice specific primer amplified not only the single exon del eted transcript but also a number of related transcripts that have deletion s in various combinations of exons. The exon 2 Delta specific primer amplif ied 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 ha ve 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 tra nscripts, 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 del etion in exon 6. The 7 Delta specific primer amplified four transcripts, th at have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 an d 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, on ly MDA-MB-435 showed the presence of exon 2 Delta and exon 4 Delta transcri pts. Analyses in the tumor samples indicated that the above transcripts are extensively modified. (C) 2000 Elsevier Science Ltd. All rights reserved.