Figure 3

RT–PCR analysis of full-length open reading frame of the WWOX cDNA and detection of aberrant transcripts in HCC cell lines. (A) PCR amplification of HCC cell line cDNAs. The 1490-bp product representing the complete open reading frame of the wild-type WWOX transcript was seen in 15 HCC cell lines, whereas only the aberrant transcripts A and B were seen in the 7703 cell line. The PLC/PRF/5 cell line showed the two smaller amplification products in addition to normal PCR product (1490 bp). The amplification of β-actin was shown as a positive control for cDNA synthesis. (B) Sequence analysis of the two aberrant WWOX transcripts in the 7703 and PLC/PRF/5 cell lines. Fragment A lacked exons 6–8 and contained an intronic DNA insertion of 96 bp. In fragment B, exons 6–8 were deleted. Arrow indicates abnormal fusion between exon 5 and exon 9. (C) DNA sequence of the newly identified WWOX splice variant. The partial sequence of WWOX exons 5 and 9 is shown and the 96-bp insertion is underlined. The predicted 32 amino-acid sequence of the novel WWOX variant is given, showing the in-frame fusion with the sequence encoded by the insertion (underlined).