[Killing effects of PWZL plasmid-mediated double suicide gene on human lens epithelium cells].
OBJECTIVE
To investigate the killing efficiency of PWZL plasmid-mediated herpes simplex virus-thymidine kinase (TK) and E. coli cytosine deaminase (CD) on human lens epithelium cells followed by the treatment of prodrugs.


METHODS
PWZL plasmid was used as a vehicle, to transduce double suicide genes into the human lens epithelium in vitro, then the cells were treated with fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell growth of the lens epithelium cells was observed by light microscope. MTT analysis was used to estimate the cell survival rate and the bystander effect was analyzed simultaneously. The significance of difference between each group was treated by statistical tests.


RESULTS
The CD and TK gene could be joined into PWZL plasmid successfully, and did not have any special effect on normal cells. There was no significant difference in cell viability between CD-TK transfected cells and control cells. Cell viability in cells treated with prodrugs was decreased in a time-dependent manner. At the end of the experiment, cell viability was lowest in GCV 10 mg/L +5-FC 60 mg/L group, GCV 10 mg/L + 5-FC 100 mg/L group and GCV 100 mg/L + 5-FC 100 mg/L group. There were no significant differences between these three groups (X2 = 1.25 , P > 0.01). Analysis of bystander effect indicated that the cell viability in GCV 100 mg/L + 5-FC 100 mg/L group and GCV 10 mg/L +5-FC 60 mg/L group was significantly lower than that in the controls (t = 10.26, 13.16; P < 0.01).


CONCLUSIONS
PWZL plasmid can transfect the CD and TK genes into lens epithelium cells successfully and efficiently. CD and TK genes can be expressed steadily. Transfection of double suicide gene reduces the dosage of prodrugs required for killing cells. The combination of 5-FC with GCV shows the greatest killing effect and also has the bystander effect.
Department of Ophthalmology, Qingdao Municipal Hospital, Qingdao 266071, China. yanxiaoran@yahoo.com.cn
Xiao-ran
XR
Yan
Hong
H
Wu
Hai-tao
HT
Yu
Xiu
X
Wang
Yu
Y
Zhang
D002470:Cell Survival; D002478:Cells, Cultured; D043525:Cytosine Deaminase / Q000235:genetics*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005437:Flucytosine / Q000494:pharmacology; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D010957:Plasmids; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D005437:Flucytosine; D013937:Thymidine Kinase; D043525:Cytosine Deaminase; D015774:Ganciclovir