Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells

Abstract

Abstract: Objective To study the killing effect of adenovirus-mediated double suicide gene under the regulation of kinase domain-containing receptor (KDR) promoter on human umbilical vein endothelial cells (HUVECs). Methods The sequences of human KDR promoter gene, CD gene and TK gene were amplified by PCR, and the plasmid pKDR-CDglyTK was constructed. A two-step transformation protocol was employed for the construction of a recombinant adenoviral plasmid pAdKDR-CDglyTK that was transfected into 293 packaging cells to further multiply and purify the adenovirus. HUVECs were infected by the resultant recombinant adenovirus of different multiplicities of infection (MOI), and the infection rate was measured by observing the expression of green fluorescence protein (GFP). The infected cells were cultured in the culture media containing ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the killing effects were evaluated. Results Recombinant adenovirus AdKDR-CDglyTK were successfully constructed, which could efficiently infect HUVEC cells, with the infection rate associated with the MOI of the recombinant adenovirus. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of the recombinant adenovirus. The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC. Conclusions Prodrug/KDR-CdglyTK system is effective in killing HUVEC cells, and its killing effect is correlated with the concentration of the prodrugs and the MOI of the recombinant adenovirus. Combination of the two prodrugs produces stronger killing effect on the cells.

CLC Number:

R394.1

HUANG Zong-hai¹, YANG Wen-yu¹, GONG Xiao-wei², QIAN Yong³, CHE Xiao-yan⁴. Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells[J]. Journal of Southern Medical University, 2004, 24(02): 139-143.

References

[1] Folkman J, Shing Y. Angiogenesis[J]. J Biol Chem, 1992, 267(16):10931-4.
[2] 范应方,黄宗海,聂晶.TNP-470对大鼠血管平滑肌细胞生长的抑制作用及细胞周期的影响[J].第一军医大学学报,2002,22(4):325-9.Fan YF, Huang ZH, Nie J. Effect of TNP-470 on rat vascular smooth muscle cell growth and cell cycle[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(4): 325-9.
[3] 贾林,袁世珍.肿瘤抗血管治疗现状与血管生成抑制-细胞毒疗法[J].广州医学院学报(J Guangzhou Med Coil),2000,28(2):82-4.
[4] 黄宗海,陈治,钱伟峰,等.血管内皮细胞生长因子及其受体在大肠癌组织中的表达[J].第一军医大学学报,2001,21(3):206-8.Huang ZH, Chen Z, Qian WF, et al. Expression of vascular endothelial growth factor and its receptor in colorectal carcinoma[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2001, 21(3): 206-8.
[5] 朱忠政,殷正丰,吴宗娣,等.腺病毒介导的KDR启动子-胸苷激酶系统对血管内皮细胞的选择性杀伤作用[J].中国肿瘤生物治疗杂志,2001,8(4):243-7.Zhu ZZ, Yin ZF, Wu ZD, et al. Adenovirus mediated gene transfer of herpes simplex virus thymidine kinase under the control of KDR promoter confers the sensitivity to ganciclovir in human vascular endothelial cells[J]. Chin J Cancer Biother, 2001, 8(4): 243-7.
[6] Mah C, Byrne BJ, Flotte TR. Virus-based gene delivery systems[J].Clin Pharmacokinet, 2002, 41(12): 901-11.
[7] Wilson DR. Viral-mediated gene transfer for cancer treatment[J].Curr Pharm Biotechnol, 2002, 3(2): 151-64.
[8] Zeng M, Smith SK, Siegel F, et al. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination[J]. Biotechniques, 2001, 31 (2): 260-2.
[9] 李颖嘉,王东.血管内皮生长因子家族与实体肿瘤[J].第一军医大学学报(J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao),2001,21(10):788-91.
[10] Yoshiji H, Gomez DE, Shibuya M, et al. Expression of vascular endothelial growth factor, its receptor, and other angioganic factors in human breast cancer[J]. Cancer Res, 1996, 56(8): 2013-6.
[11] Folkman J. The role of angiogenesis in tumor growth[J]. Semin Cancer Biol, 1992, 3: 65-71.
[12] 钱伟峰,黄宗海.CD-TK融合自杀基因治疗肿瘤的研究[J].实用癌症杂志(J Pract Cancer),2000,15(1):103-4.