HRE Enhance Killing Effect Of HSV-TK/GCV To Ewing Sarcoma Cells Under Hypoxic Condition

Objective: The hypoxic cells in solid tumours were resistant to both radiotherapy and chemotherapy. To improve the effectiveness of radiotherapy and chemotherapy to these tumours, a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed. HRE enhancing killing effect of HSV-TK/gancyclovir (GCV) to Ewing sarcoma cell line SK-ES-1 was observed under hypoxia condition.Methods: The HRE was synthesized according to the literature and cloned into the enhancer site of pERES2-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the Mutiple Clone Site of pIRES2-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing sarcoma cell line SK-ES-1 was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxia (21% oxygen) or hypoxia (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing sarcoma cell line SK-ES-1 transfected with pTK or pHRE-TK recombinant plasmid to the gancyclovir (GCV) was determined with method of tetrazolium (MTT) after treated with GCV for five days.Result:1. The sequencing result showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction.2. Comparing study of fluorescent optical density (FOD) showed: (1). The EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia, p<0.01. (2). When exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group, p<0.01. (3). There was not any difference among the four group cells when exposed to normoxia.3. Comparing study of the sensitivity of three group cells to GCV showed: (1). The pHRE-TK and pTK group cells were much more sensitive to GCV than pHRE group under hypoxia condition, p<0.01. The higher the GCV concentration, the more the difference.. (2). The pHRE-TK group cells were more sensitive to GCV than pTK group under hypoxia condition, p<0.01, but were almost same sensitive under normoxia condition. (3). The pHRE-TK group cells had more sensitivity to GCV under hypoxia than normoxia, p<0.01. The pTK group cells had almost same sensitivity to GCV under hypoxia and normoxia.Conclusion:1. The eukaryotic expression vector expressing HSV-TK regulated by HRE under hypoxia was constructed successfully.2. HRE could enhance killing effect of HSV-TK/GCV to human Ewing sarcoma cell line under hypoxia condition.