A B S T R A C TUsing GGCX gene-specific real-time PCR, exon 2 deletion splice variant of vitamin K-dependent g-glutamyl carboxylase (GGCX) mRNA was identified in HCC cell lines. Expressions of wild type and exon 2 deletion variant of GGCX were analyzed with relevance to DCP production in HCC cell lines. Hep3B, HepG2, HuH1, HuH7, and PLC/PRF/5 produced DCP, while SK-Hep-1, HLE, HLF, and JHH1 produced no detectable level of DCP. DCP-producing cells expressed exon 2 deletion variant of GGCX mRNA and protein, while DCP-negative cells expressed no detectable level of exon 2 deletion variant of GGCX. These results suggest that exon 2 deletion splice variant of GGCX causes dysfunction of GGCX enzyme activity resulting in DCP production in HCC cell lines. Published by Elsevier B.V. All rights reserved.
IntroductionHepatocellular carcinoma (HCC) accounts for more than 80% of all primary liver cancers and is one of the most common malignancies worldwide (Kassahun et al., 2006;Okuda et al., 2000;Parkin et al., 1999). Since advanced HCC has a poor prognosis, early detection is important to improve prognosis of HCC patient. Liver ultrasonography and monitoring of tumor markers are helpful for early detection of HCC. There are two major tumor markers for HCC: a-fetoprotein (AFP) andAbbreviations: DCP, des-g-carboxy prothrombin; HCC, hepatocellular carcinoma; GGCX, vitamin K-dependent g-glutamyl carboxylase; AFP, a-fetoprotein; VKOR, vitamin K epoxide reductase; D2GGCX, exon 2 deletion splice variant of GGCX; PLC, PLC/PRF/5; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ECLIA, electrochemiluminescence immunoassay; TBS-T, TBS with Tween-20; WT, wild type; PCR, polymerase chain reaction; RT-PCR, real-time quantitative PCR; ACTB, b-actin; CMVp, cytomegalovirus promoter; GFP, green fluorescent protein; ESE, exonic splicing enhancer.* Corresponding author.