All the cells were transfected with MK2.3-TK, AFPEn0.2-TK or vector DNA and then respective G418-resistant cells were exposed to various concentrations of GCV (Figure 3). The sensitivity of vector DNA-transfected cells was not different from that of respective parent cells. However, AFP-producing HuH-7 and PLC/PRF/5 cells increased selleck chem Rapamycin the susceptibility to GCV, when they were transfected with MK2.3- or AFPEn0.2-TK DNA (Figure 3A and B). PLC/PRF/5 cells were more susceptible to GCV than HuH-7 cells, although the transcriptional activity of both MK and AFP promoters was stronger in HuH-7 than in PLC/PRF/5 cells. AFP-nonproducing HCC and non-HCC cells that were transfected with MK2.3-TK DNA significantly increased their susceptibility to GCV and those transfected with AFPEn0.

2-TK DNA also slightly increased the susceptibility (Figure 3C�CF). The increased sensitivity of the AFPEn0.2-TK transfectants, despite minimal transcriptional activity of the AFPEn0.2 DNA in these cells, was probably due to multiple integration of transfected DNA, in which condition cellular and/or plasmid-derived promoters could activate the TK gene. Figure 3 Susceptibility of HuH-7 (A), PLC/PRF/5 (B), HLE (C), HLF (D), MCF-7 (E) and AsPC-1 (F) cells to various concentrations of GCV. Parent cells and G418-resistant cells that were transfected with vector, MK2.3-TK or AFPEn0.2-TK DNA were used. … DISCUSSION In this study, we showed that human HCC specimens expressed the MK gene more frequently than the AFP gene and that the transcriptional activity of the MK promoter was as strong as that of the enhancer-linked AFP promoter.

The present data also indicate that the MK promoter could activate the HSV-TK gene in HCC cells to a similar extent as the AFP promoter. The AFP promoter has been used to activate a suicide gene in HCC cell lines (Kanai et al, 1996; Mawatari et al, 1998), to produce a hybrid promoter in combination with other regulatory regions (Ido et al, 2001) and to apply to oncolytic adenovirus (Hallenbeck et al, 1999). These previous studies suggested the feasibility of the AFP promoter-mediated gene therapy for HCC. However, relatively low frequency of AFP expression in human HCC may hamper the clinical application. Although serum AFP was elevated in 70% of HCC patients (Nakabayashi et al, 1991), AFP transcripts were not frequently detected with Northern blot analysis as shown in the present study.

Carfilzomib Di Biscegile et al. (1986) reported that nine out of 10 HCC patients showed elevated serum AFP values, but only two out of the 10 HCC specimens were positive for the mRNA with Northern blot analysis. Similar cases, high serum AFP values with undetectable AFP transcript with Northern blot analysis, were also reported (Varagona et al, 1992; Niwa et al, 1996), although the level of AFP mRNA expression in general correlated with serum AFP levels (Niwa et al, 1996).