HER-2/neu is a potent oncogene that predicts poor outcome when overexpressed in ovarian cancer. The SKOV-3 ovarian carcinoma cell line, one of the only models for HER2-driven ovarian cancer, expresses a major uncharacterized 8-kb alternative HER-2 transcript.
Objectives: The aim of this study was to characterize the structure and determine the origin of the alternative sequence and examine the possible role of the 8-kb alternative transcript in overexpression of the HER-2 gene.
Methods: The structure of the 8-kb transcript was investigated using polymerase chain reaction (PCR) and nucleotide sequencing of cDNA clones. PCR analysis of genomic DNA was used to assess the origin of the 8-kb transcript. The stability of the 8-kb mRNA was assessed by Northern blot analysis of RNA extracted from cells treated with transcriptional inhibitors.
Results: Similar 5'UTR and coding sequence but an extended 3'UTR were contained in the 8-kb compared to the well-characterized 4.5-kb HER-2 transcript. Genomic DNA had continuity between the novel 3'UTR sequence from the 8-kb transcript and adjacent HER-2 terminal exon sequence. The 8-kb transcript had a half-life of 13 h compared to 5.5 h for the 4.5-kb transcript (P<0.01).
Conclusions: The 8-kb transcript is generated from alternative polyadenylation site usage rather than gene rearrangement. Since the 8-kb transcript contains alternative sequence found at the 3' end of the normal HER-2 gene, it could be expressed in other cells. Increased stability of the 8-kb transcript may confer a selective advantage for SKOV-3 cells by providing enhanced HER-2 expression.
Copyright 1999 Academic Press.