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Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo

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Abstract

Neuroblastoma arises as a direct result of genetic disorder; therefore, it should be well treated and conquered by gene therapy in future. In this study, neuroblastoma cell line SH-SY5Y experiments, in vitro and in nude mice in vivo, were subjected to research thymidine kinase suicide gene to treat neuroblastoma. The plasmid LXpsp-hytk and a plasmid LXSH were transduced separately by lipofectin into human neuroblastoma cell line SH-SY5Y. SH-SY5Y-hy and SH-SY5Y-hytk were selected by hygromycin B. Different ganciclovir (GCV) concentration was given to SH-SY5Y-hytk to determine optimal GCV concentration. The cytotoxic effect of GCV on SH-SY5Y-hytk, SH-SY5Y-hy, and SH-SY5Y cells was determined. Scapular subcutaneous tumors were established in nude mice by inoculating 2.5×106 SH-SY5Y-hytk on their left sides and 2.5×106 SH-SY5Y-hy cells on their right sides for every mouse of treatment group and control group, respectively. After 1 week, mass grew in both sides of all the mice, and from the eighth day on, every mouse in treatment group received daily intraperitoneal injection of GCV 50 mg/kg body weight for 14 days; every mouse in control group received daily intraperitoneal injection of 1 ml saline for 14 days. On day 22 tumors were excised and weighed on the left and right sides, respectively, and apoptosis was detected by TUNEL method. Apoptotic index was calculated on the left and on the right sides, respectively, for every mouse in treatment group and control group. The lowest concentration of hygromycin B was 60 μg/ml. The cytotoxic effect of GCV on SH-SY5Y-hytk cells was obvious (IC50=0.03 μM), whereas GCV showed almost no cytotoxic effect on SH-SY5Y and SH-SY5Y-hy cells (IC50>400 μM). SH-SY5Y-hytk was killed by concentrations of 30 μM GCV effectively and it obviously showed the bystander effect, when SH-SY5Y-hytk remained at least 18% in the mixture culture cells. The tumor on the left side was much smaller than that of the right side in control group (p<0.05), and apoptotic index of the left was higher than that of the right in control group (p<0.01). SH-SY5Y-hytk has the bystander effect over 18% SH-SY5Y-hytk of the mixture culture cells at the concentration of 30 μM GCV. The HSV-tk/GCV system was effective in treating SH-SY5Y neuroblastoma cell line in vivo as well. Our findings suggest that thymidine kinase gene therapy could be a potential method for treating neuroblastoma in the future.

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References

  1. Boucher PD, Ostruszka LJ, Murphy PJ, Shewach DS (2002) Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy. Gene Ther 9:1023–1030

    Article  CAS  PubMed  Google Scholar 

  2. Sangro B, Qian C, Schmitz V et al. (2002) Gene therapy of hepatocellular carcinoma and gastrointestinal tumors. Ann N Y Acad Sci 963:6–12

    CAS  PubMed  Google Scholar 

  3. Wu Q, Moyana T, Xiang J (2001) Cancer gene therapy by adenovirus-mediated gene transfer. Curr Gene Ther 1:101–122

    CAS  PubMed  Google Scholar 

  4. Akatsuka Y, Emi N, Kato H et al. (1994) Retrovirus-mediated transfer of a hygromycin B phospho transferase thymidine kinase fusion gene into human CD34+ bone marrow cells. Int J Hematol 60:251–258

    CAS  PubMed  Google Scholar 

  5. Lupton SD, Brun LL, Kalberg VA (1991) Dominant positive and negative selection using a hygromycin B phosphotransferase-thymidine kinase fusion gene. Mol Cell Biol 11:3374–3379

    CAS  PubMed  Google Scholar 

  6. Karara AL, Bumaschny VF (2002) Lipofection of early passages of cell cultures derived from murine adenocarcinomas: in vitro and ex vivo testing of the thymidine kinase/ganciclovir system. Cancer Gene Ther 9:96–99

    Article  CAS  PubMed  Google Scholar 

  7. Su H, Lu R, Chang JC et al. (1997) Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice. Proc Natl Acad Sci USA 94:13891–13896

    Article  CAS  PubMed  Google Scholar 

  8. Haberkorn U, Khazaie K, Morr L et al. (1998) Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase. Nucl Med Biol 25:367–373

    CAS  PubMed  Google Scholar 

  9. Tomicic M, Bey E, Wutzler P et al. (2002) Comparative analysis of DNA breakage, chromosomal aberrations and apoptosis induced by the anti-herpes purine nucleoside analogues aciclovir, ganciclovir and penciclovir. Mutat Res 505:1–11

    Article  CAS  PubMed  Google Scholar 

  10. Tomicic MT, Thust R, Kaina B (2002) Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation. Oncogene 28:2141–2153

    Article  Google Scholar 

  11. Nishihara E, Nagayama Y, Mawatari F et al. (1997) Retrovirus-mediated herpes simplex virus thymidine kinase gene transduction renders human thyroid carcinoma cell lines sensitive to ganciclovir and radiation in vitro and in vivo. Endocrinology 138:4577–4583

    CAS  PubMed  Google Scholar 

  12. Lechanteur C, Princen F, Lo Bue S et al. (1997) HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis. Gene Ther 4:1189–1194

    CAS  PubMed  Google Scholar 

  13. Namba H, Tagawa M, Lwadate Y et al. (1998) Bystander effect-mediated therapy of experimental brain tumor by genetically engineered tumor cells. Hum Gene Ther 9:5–11

    Google Scholar 

  14. Beltinger C, Fulda S, Walczak H et al. (2002) TRAIL enhances thymidine kinase/ganciclovir gene therapy of neuroblastoma cells. Cancer Gene Ther 9:372–381

    Article  CAS  PubMed  Google Scholar 

  15. Burrows FJ, Gore M, Smiley WR et al. (2002) Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transduced tumor cells. Cancer Gene Ther 9:87–95

    Article  CAS  PubMed  Google Scholar 

  16. Touraine RL, Ishii-Morita H, Ramsey WJ et al. (1998) The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication. Gene Ther 5:1705–1811

    Article  CAS  PubMed  Google Scholar 

  17. Fukunaga M, Takamori S, Hayashi A et al. (2002) Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model. Ann Thorac Surg 73:1740–1746

    Article  PubMed  Google Scholar 

  18. Panis Y, Caruso M, Houssin D et al. (1992) Treatment of experimental liver tumors by in vivo suicide gene transfer in rats. CR Acad Sci 315:541–544

    CAS  Google Scholar 

  19. Warren P, Song W, Holle E (2002) Combined HSV-TK/GCV and secondary lymphoid tissue chemokine gene therapy inhibits tumor growth and elicits potent antitumor CTL response in tumor-bearing mice. Anticancer Res 22:599–604

    CAS  PubMed  Google Scholar 

  20. Puhlmann M, Brown CK, Gnant M et al. (2000) Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. Cancer Gene Ther 7:66–73

    Article  CAS  PubMed  Google Scholar 

  21. Sa Cunha A, Bonte E, Dubois S et al. (2002) Inhibition of rat hepatocellular carcinoma tumor growth after multiple infusions of recombinant Ad.AFPtk followed by ganciclovir treatment. J Hepatol 37:222–230

    Article  PubMed  Google Scholar 

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  1. Jing-Zhe Zhang

    Present address: Department of Pediatric Surgery, Beijing Children's Hospital, 100020, Beijing, China

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  1. Capital Institute of Pediatrics, 100020, Beijing, China

    Xun Bi & Jing-Zhe Zhang

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  1. Xun Bi
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Bi, X., Zhang, JZ. Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo. Ped Surgery Int 19, 400–405 (2003). https://doi.org/10.1007/s00383-003-1019-0

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  • DOI: https://doi.org/10.1007/s00383-003-1019-0

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