Abnormal splicing of primary RNA transcripts of normal genes is a recognized mechanism for the production of some abnormal proteins found in cancer cells. A misspliced form of the cholecystokinin-B/gastrin (CCK-B) receptor recently was reported to be present in colon carcinoma, where it was postulated to play a role in stimulating tumor growth (M. R. Hellmich et al, J. Biol. Chem., 275: 32122-32128, 2000). Here, we report the presence of the same abnormal protein in pancreatic carcinoma and explore the molecular basis for this missplicing event. Reverse transcription-PCR and sequencing were used to demonstrate the presence of a misspliced form of the CCK-B receptor having its fourth intron retained in three pancreatic cancer cell lines and in tumor tissue, but not in surrounding healthy pancreas, from two patients with pancreatic carcinoma. A mini-gene construct representing the region of this gene from its third through its fifth exon and containing the two intervening introns was produced and transiently expressed in the MIA PaCa-2 human pancreatic cancer cell line. Specific reverse transcription-PCR reactions with both vector-derived and receptor-specific primers demonstrated the presence of both correctly fully spliced and selectively misspliced forms of this receptor. Mutagenesis of the mini-gene demonstrated that a suboptimal sequence at the 3′-end of intron 4 contributed to this missplicing. This focused attention on the U2 small nuclear ribonucleoprotein particle auxiliary splicing factors (U2AFs) known to interact specifically with this domain. Indeed, quantitative real-time PCR demonstrated a reduced level of expression of one of these factors, U2AF35, in pancreatic cancer cells compared with healthy pancreas. Furthermore, the relative amount of missplicing of the CCK-B receptor mini-gene in the pancreatic cancer cell line was reversed by transfection of the cells with U2AF35 cDNA. This work describes the presence of an additional abnormal protein in pancreatic cancer and describes a new molecular mechanism for its production, providing additional potential therapeutic targets.
|Original language||English (US)|
|Number of pages||6|
|State||Published - Feb 1 2002|
ASJC Scopus subject areas
- Cancer Research