Establishment and Research of Human Hepatocellular Carcinoma Cell Line Stably Transfected with Linamarase_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LI Jun ¹ , ZHU Liang ² , ZHENG Ruiguo ³ , LI Haimin ³ , CHEN Minhao ³ , MA Jun ³ , DOU Kefeng. ³

1. Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China;;
2.Department of Medical Education, The Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China;;
3.Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China;

Corresponding author：

Keywords：

Linamarase; Suicide gene; Enzyme-prodrug system; Hepatocellular carcinoma

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ObjectiveTo construct a hepatocellular carcinoma (HCC) cell line stably transfected with linamarase (lis) gene from cassava, and to study its biological characteristics. MethodsLis DNA was amplified from cassava by PCR and cloned into the pcDNA3.1(+) plasmid. Then the recombinant plasmid pcDNA3.1/lis was transfected into human HCC HepG2 cell line using lipofectamineTM 2000 and G418 selection. The stably transfected cell lines HepG2/lis was identified by immunofluorescence, RT-PCR, and Western blot. The enzyme activity of lis in cells was assayed by Lambert method. The characteristics of the new cell lines were checked by several methods: the cell growth curve was observed by MTT, cell cycle was analyzed by flow cytometry, and characteristics of tumor formation in vivo were detected in nude mice. ResultsLis from the cassava could stably integrate into eukaryotic cells, and package the same protein as lis with β-glucosidase activity. The stable integration of lis in cells did not interfere with cell morphology, growth characteristics, cell cycle, and tumorigenesis in vivo significantly. ConclusionsA new HCC cell lines transfected with lis is successfully established, which may lay an experimental foundation for the study of HCC treatment by using lis suicide gene system in future.

Citation： LI Jun,ZHU Liang,ZHENG Ruiguo,LI Haimin,CHEN Minhao,MA Jun,DOU Kefeng.. Establishment and Research of Human Hepatocellular Carcinoma Cell Line Stably Transfected with Linamarase. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2011, 18(8): 829-834. doi: Copy

1.	Santana MA, Vásquez V, Matehus J, et al. Linamarase expression in cassava cultivars with roots of low and highcyanide content [J]. Plant Physiol, 2002, 129(4): 16861694.
2.	ItohNashida T, Hiraiwa M, Uda Y. Purification and properties of betaDglucosidase (linamarase) from the butter bean, Phaseolus lunatus [J]. J Biochem, 1987, 101(4): 847854.
3.	Link N, Aubel C, Kelm JM, et al. Therapeutic protein transduction of mammalian cells and mice by nucleic acidfree lentiviral nanoparticles [J]. Nucleic Acids Res, 2006, 34(2): e16.
4.	GarcíaEscudero V, Gargini R, Izquierdo M. Glioma regression in vitro and in vivo by a suicide combined treatment [J]. Mol Cancer Res, 2008, 6(3): 407417.
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6.	Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55(2): 74108.
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11.	Huang X, Zhang W, Wakimoto H, et al. Adenovirusmediated tissuespecific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression levels [J]. J Exp Ther Oncol, 2002, 2(2): 100106.
12.	李锋, 张萍, 王楚, 等. PNP/fludarabine自杀基因系统对人肝癌细胞HepG2体外杀伤效应 [J]. 中国肿瘤, 2004, 13(12): 800805.
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15.	Cool V, Pirotte B, Gérard C, et al. Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with 9L gliosarcoma [J]. Hum Gene Ther, 1996, 7(5): 627635.
16.	Cortes ML, de Felipe P, Martin V, et al. Successful use of a plant gene in the treatment of cancer in vivo [J]. Gene Ther, 1998, 5(11): 14991507.
17.	Kousparou CA, Epenetos AA, Deonarain MP. Antibodyguided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells [J]. Int J Cancer, 2002, 99(1): 138148.
18.	Birchall JC, Kellaway IW, Gumbleton M. Physical stability and in vitro gene expression efficiency of nebulised lipidpeptideDNA complexes [J]. Int J Pharm, 2000, 197(12): 221231.
19.	卢银平, 王宝菊, 黄红平, 等. 中国旱獭干扰素α家族基因在真核细胞和原核细胞中的表达 [J]. 中华肝脏病杂志, 2006, 14(2): 124128.
20.	Thompson L. Gene therapy. Monkey tests spark safety review [J]. Science, 1992, 257(5078): 1854.
21.	代文杰, 姜洪池. 肝细胞基因转导载体系统应用进展 [J]. 中国普外基础与临床杂志, 2002, 9(1): 5860.
22.	Hernández T, Lundquist P, Oliveira L, et al. Fate in humans of dietary intake of cyanogenic glycosides from roots of sweet cassava consumed in Cuba [J]. Nat Toxins, 1995, 3(2): 114117.
23.	Frakes RA, Sharma RP, Willhite CC. Comparative metabolism of linamarin and amygdalin in hamsters [J]. Food Chem Toxicol, 1986, 24(5): 417420.
24.	Cortés ML, GarcíaEscudero V, Hughes M, et al. Cyanide bystander effect of the linamarase/linamarin killersuicide gene therapy system [J]. J Gene Med, 2002, 4(4): 407414.
25.	胡春松, 洪均言, Losordo DW. 基因治疗的安全性、有效性和稳定性原则 [J]. 中国实验血液学杂志, 2004, 12(3): 392396.

1. Santana MA, Vásquez V, Matehus J, et al. Linamarase expression in cassava cultivars with roots of low and highcyanide content [J]. Plant Physiol, 2002, 129(4): 16861694.
2. ItohNashida T, Hiraiwa M, Uda Y. Purification and properties of betaDglucosidase (linamarase) from the butter bean, Phaseolus lunatus [J]. J Biochem, 1987, 101(4): 847854.
3. Link N, Aubel C, Kelm JM, et al. Therapeutic protein transduction of mammalian cells and mice by nucleic acidfree lentiviral nanoparticles [J]. Nucleic Acids Res, 2006, 34(2): e16.
4. GarcíaEscudero V, Gargini R, Izquierdo M. Glioma regression in vitro and in vivo by a suicide combined treatment [J]. Mol Cancer Res, 2008, 6(3): 407417.
5. Lambert JL, Ramasamy J, Paukstelis JV. Stable reagents for the colorimetric determination of cyanide by modified Koenig reactions [J]. Anal Chem, 1975, 47 (6): 916918.
6. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55(2): 74108.
7. 沈志勇, 刘骅, 曹晖. 胃癌基因诊断和治疗的研究进展 [J]. 中国普外基础与临床杂志, 2009, 16(6): 500502.
8. 任涛, 李枚娟, 颜江华. 自杀基因治疗恶性肿瘤的研究现状及展望 [J]. 现代肿瘤医学, 2009, 17(12): 24352437.
9. 李越. 肝癌的自杀基因治疗的研究进展 [J]. 医学理论与实践, 2008, 21(1): 3940.
10. Sung MW, Yeh HC, Thung SN, et al. Intratumoral adenovirusmediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma: results of a phase I clinical trial [J]. Mol Ther, 2001, 4(3): 182191.
11. Huang X, Zhang W, Wakimoto H, et al. Adenovirusmediated tissuespecific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression levels [J]. J Exp Ther Oncol, 2002, 2(2): 100106.
12. 李锋, 张萍, 王楚, 等. PNP/fludarabine自杀基因系统对人肝癌细胞HepG2体外杀伤效应 [J]. 中国肿瘤, 2004, 13(12): 800805.
13. Huber BE, Richards CA. Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virusdirected enzyme prodrug therapy (VDEPT) and other gene therapy applications [J]. J Drug Target, 1996, 3(5): 349356.
14. 陈耿臻，韩慧，许铭炎，等. ADVtk重组腺病毒的构建及检测 [J]. 中国普外基础与临床杂志， 2010， 17（7）： 703706.
15. Cool V, Pirotte B, Gérard C, et al. Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with 9L gliosarcoma [J]. Hum Gene Ther, 1996, 7(5): 627635.
16. Cortes ML, de Felipe P, Martin V, et al. Successful use of a plant gene in the treatment of cancer in vivo [J]. Gene Ther, 1998, 5(11): 14991507.
17. Kousparou CA, Epenetos AA, Deonarain MP. Antibodyguided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells [J]. Int J Cancer, 2002, 99(1): 138148.
18. Birchall JC, Kellaway IW, Gumbleton M. Physical stability and in vitro gene expression efficiency of nebulised lipidpeptideDNA complexes [J]. Int J Pharm, 2000, 197(12): 221231.
19. 卢银平, 王宝菊, 黄红平, 等. 中国旱獭干扰素α家族基因在真核细胞和原核细胞中的表达 [J]. 中华肝脏病杂志, 2006, 14(2): 124128.
20. Thompson L. Gene therapy. Monkey tests spark safety review [J]. Science, 1992, 257(5078): 1854.
21. 代文杰, 姜洪池. 肝细胞基因转导载体系统应用进展 [J]. 中国普外基础与临床杂志, 2002, 9(1): 5860.
22. Hernández T, Lundquist P, Oliveira L, et al. Fate in humans of dietary intake of cyanogenic glycosides from roots of sweet cassava consumed in Cuba [J]. Nat Toxins, 1995, 3(2): 114117.
23. Frakes RA, Sharma RP, Willhite CC. Comparative metabolism of linamarin and amygdalin in hamsters [J]. Food Chem Toxicol, 1986, 24(5): 417420.
24. Cortés ML, GarcíaEscudero V, Hughes M, et al. Cyanide bystander effect of the linamarase/linamarin killersuicide gene therapy system [J]. J Gene Med, 2002, 4(4): 407414.
25. 胡春松, 洪均言, Losordo DW. 基因治疗的安全性、有效性和稳定性原则 [J]. 中国实验血液学杂志, 2004, 12(3): 392396.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Establishment and Research of Human Hepatocellular Carcinoma Cell Line Stably Transfected with Linamarase

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