|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
In this research, enhancement of bystander effects with the combination of immuno-gene therapy and pro-drug gene therapy was analyzed. As a main portion of this project, effects of UPRT (uracil phosphoribosyltransferase) gene therapy (new type of pro-drug gene therapy) for bladder cancer was analyzed. Here we show the summary of them.
Objective : We evaluated whether adenovirus-mediated UPRT genes could generate sensitized antitumor effect of 5-FU on experimental bladder cancer.
Material and Method : Human bladder cancer cell T24 was used for in vitro and in vivo study. Two recombinant replication-deficient adeno virus vectors, AdCA-LacZ and AdCA-UPRT, were constructed from a serotype 5 wild-type adenovirus (Ad5) by inserting CAG promoter with an E. coli LacZ gene and an E. coli UPRT gene, respectively, into the EIA region of the genome, deleting the E1B and E3 region. For in vitro chemosensitivity assay, T24 cells were seeded in 96-well plates at a density of 1000 cells/well, infected w
ith AdCA-LacZ or AdCA-UPRT (MOI :0-100), and then exposed to 5-FU. Six days later, the sensitivity was assessed with MTT assay. For in vivo tumor treatment, subcutaneous tumors were employed by injection of T24 cells (10^7 cells). Twelve days after tumor cell injection (day 12) when tumor becomes palpable with mean estimated weight 147mg (108 -199mg), mouse were randomized to be directly injected with AdCA-LacZ or AdC A-UPRT (1X10^8 pfu/10ul) subsequently (two days later : day14) with either saline or 5-FU(2Omg/kg) intraperitoneally for 10days (from day14 to 23).
Results : AdCA-UPRT infection increased the sensitivity of T24 cells to 5-FU in a MOI-dependent manner. IC50 to 5-FU shifted from 5,М in, control cells to 0.05М in AdCA-UPRT-infected cells at MOI of 50 indicating 100 fold sensitization by AdCA-UPRT infection. Consistent with in vitro studies, administration of AdCA-UPRT into the tumor of T24 cells, together with systemic 5-FU administration, resulted in sup pression of tumor growth at day28, compared with the control tumor groups : Among control groups, difference was not observed in comparis on with the presence or absence of 5-FU administration
Conclusion : These results suggest that UPRT gene therapy with 5-FU can sensitize the antitumor effect of 5-FU. Consequently, this approach is a new chemosensitizing strategy for cancer gene therapy and a more feasible modality for the treatment of bladder cancer. Less