THE INFLUENCE OF HUMAN SINGLE CHAIN INTELEUKIN-12 GENE TRANSDUCTION ON THE BIOLOGICAL BEHAVIOR OF HEPATOMA 7721 CELLS

Objective. To investigate the anti-tumor effects of human single chain interleukin-12 (hscIL-12). Method. pcDNA/hscIL-12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL-12 cells which secrete hscIL-12 stably, were obtained via G418 selection, and in vitro the influence of hscIL-12 gene transduction on the growth of tumor cells was evaluated by cellcycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL-12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice, respectively. 7721/pcDNA and 7721/hscIL-12 groups were divided into two sub-groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equalvolume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL-12 produced stably by 7721/hscIL-12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the7721/hscIL-12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL-12 cells. In ani-mal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5 -7mm,while those of 7721/hscIL-12 group were 2 -4mm. When treated with hPBL, the tumor of 7721/hscIL-12 groupdisappeared completely. Histologically, the tumors from 7721/hscIL-12 without hPBL treatment had numerouslymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis, whereas the tumors of 7721 and 772l/pcDNA groups grew thrivingly.Conclusion. hsclL-12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL-12 has certain effects on mice immune system. These findings suggest that hscIL-12 and hscIL-12 gene therapy might have promising prospects in clinical application.

THE INFLUENCE OF HUMAN SINGLE CHAIN INTELEUKIN- 12 GENE TRANSDUCTION ON THE BIOLOGICAL BEHAVIOR OF HEPATOMA 7721 CELLS

OBJECTIVE: To investigate the anti-tumor effects of human single chain interleukin-12 (hscIL-12). METHOD: pcDNA/hscIL-12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL-12 cells which secrete hscIL-12 stably, were obtained via G418 selection, and in vitro the influence of hscIL-12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL-12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice, respectively. 7721/pcDNA and 7721/hscIL-12 groups were divided into two sub-groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. RESULTS: hscIL-12 produced stably by 7721/hscIL-12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL-12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL-12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5 approximately 7 mm, while those of 7721/hscIL-12 group were 2 approximately 4 mm. When treated with hPBL, the tumor of 7721/hscIL-12 group disappeared completely. Histologically, the tumors from 7721/hscIL-12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis, whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly. CONCLUSION: hscIL-12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL-12 has certain effects on mice immune system. These findings suggest that hscIL-12 and hscIL-12 gene therapy might have promising prospects in clinical application.