Regulated expression of a suicide gene with the promoters can primarily destroy tumour cells and leave the surrounding now tissues undamaged. The ��-fetoprotein promoter (AFP) is a representative one to activate an exogenous gene specifically in hepatocellular carcinoma (HCC) (Tamaoki, 2000) and has been applied to targeted gene therapy for HCC (Kanai et al, 1996; Mawatari et al, 1998; Hallenbeck et al, 1999). Precise analysis of the regulatory region of the AFP gene demonstrated that enhancer and silencer elements in the region played a crucial role in the transcription (Nakabayashi et al, 1991) and the silencer-deleted, enhancer-linked AFP promoter showed noticeable therapeutic effects compared with the unmodified AFP promoter (Mawatari et al, 1998).
Midkine (MK) is a heparin-binding growth factor (Kadomatsu et al, 1988) with a number of functions: forced expression of MK induced transformation (Kadomatsu et al, 1997) and elevated angiogenesis (Choudhuri et al, 1997). Expression of the MK gene in human adult tissues is extremely low and restricted; however, the expression is upregulated in a number of human tumours particularly in gastrointestinal tumours including HCC (Aridome et al, 1995; Koide et al, 1999). The 5�� upstream region of the MK gene was demonstrated to activate a suicide gene in MK-positive human tumours (Adachi et al, 2000; Miyauchi et al, 2001; Wesseling et al, 2001). In this study, we compared the usefulness of the AFP and the MK promoters for HCC treatment by examining the frequency of MK and AFP expression in human HCC specimens and the transcriptional activity of the promoters in HCC cell lines.
MATERIALS AND METHODS Tissue samples and cells Paired cancerous and noncancerous specimens obtained from the same HCC patients were provided by the Chiba Cancer Center Tissue Bank. The clinical data regarding respective patients were provided by the Bank based on the ethical guideline on human genome study and genetic Anacetrapib analysis (issued by the Japanese government in 2001). Histological types of HCC were determined according to the classification of the Liver Cancer Study Group of Japan. Human HCC cells (HuH-7, PLC/PRF/5, HLE and HLF cells), human breast cancer MCF-7 cells and human pancreatic cancer AsPC-1 cells were cultured in Dulbecco’s modified Eagle’s or RPMI1640 medium supplemented with 10% fetal calf serum. Northern blot analysis Total RNA (10��g) was isolated with Trizol (Life Technologies, Rockville, MD, USA), subjected to electrophoresis in a denaturing formaldehyde�Cagarose gel and transferred to a nylon filter. MK (Kadomatsu et al, 1988) and AFP cDNA (Morinaga et al, 1983) were labelled with [32P]dCTP and used as probes.