1% SDS/0.1mgml?1 salmon sperm DNA at 42��C for 12h. The filter was washed with a solution of 0.2 �� SSPE/0.1% SDS several blog post times at 50��C. The same filter was rehybridised with a human 18S ribosome cDNA as a control. Dual luciferase assay The 2335-bp genomic DNA fragment of the MK gene (?2285/+50, +1 corresponds to the transcription start site) (Uehara et al, 1992) was cloned into the pGL2-basic vector (Promega, Madison, WI, USA) that contained the firefly luciferase gene without any promoter sequences (MK2.3-luc). MK2.3kb-luc DNA was digested with XhoI/Eco47III to produce the 609-bp fragment-linked firefly luciferase genes (MK0.6-luc). The AFP promoter region (AFP0.2, 212-bp SacI/HindIII fragment) and the enhancer (934-bp SwaI/EcoRI fragment)-linked AFP promoter region (AFPEn0.

2) were prepared from pAF5.1-CAT (Nakabayashi et al, 1991), and they were subcloned into pGL2-basic vector (AFP0.2-luc, AFPEn0.2-luc). The transcriptional activity was measured with the dual luciferase reporter assay system (Promega). Plasmid DNA containing respective genomic fragments, pGL2-control vector (Promega) harbouring the SV40 promoter-linked firefly luciferase gene (SV40-luc) or pGL2-basic, together with a control vector, the renilla luciferase gene fused with the HSV-TK promoter (pRL-TK, Promega) at a molar ratio of 10:1, was transfected into target cells with a lipofectin reagent (Life Technologies, Gaithersburg, MD, USA). After 2 days, cells were lysed and the luciferase activities were measured according to the manufacturer’s protocol.

The relative firefly luciferase activity of each cell lysate was calculated based on the amount of luminescence produced by renilla luciferase and was expressed as a percentage of the SV40 promoter-mediated activity. All of the values were expressed as a mean of four independent experiments. The statistical analysis was performed by one-way analysis of variance (ANOVA). In vitro sensitivity to ganciclovir The 2.3kb. MK or the enhancer-linked AFP promoter region was ligated into pcDNA3 vector (Invitrogen, San Diego, CA, USA) from which the cytomegalovirus promoter was removed. The herpes simplex virus-thymidine kinase (HSV-TK) gene was then cloned into the downstream of the promoters (MK2.3-TK, AFPEn0.2-TK). Cells were transfected with MK2.3-TK, AFPEn0.2-TK or pcDNA3 vector DNA and G418 (Life Technologies)-resistant cells were selected.

The pooled cells were seeded in 96-well plates at a density of 5 �� 103cellswell?1 and were cultured with various concentrations of ganciclovir (GCV). After 5 days, viable cells in each well were measured with a cell-counting kit (Wako, Osaka, Japan). The amount of formazan produced in each well was AV-951 determined from the absorbance at 450nm. RESULTS Expression of the MK and AFP genes in HCC We examined the frequency of expressed MK and AFP genes in paired HCC and noncancerous specimens of the same patients by Northern blot analysis.