TY  - JOUR
PY  - 2004//
TI  - Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells
JO  - Chinese medical journal
A1  - Cao, Hui-qing
A1  - Meng, Xian-min
A1  - Liu, Dong-qing
A1  - Zhao, Xiu-wen
A1  - Ding, Jin-feng
SP  - 1464
EP  - 1470
VL  - 
IS  - 24
N2  - &lt;p&gt;&lt;b&gt;BACKGROUND&lt;/b&gt;Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;METHODS&lt;/b&gt;Adenoviral vectors, including Ad-EF1alpha-CD-cytomegalovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1alpha-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA + E. coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis.&lt;/p&gt;&lt;p&gt;&lt;b&gt;RESULTS&lt;/b&gt;Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1alpha-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1alpha-CD single gene groups. The rate of cell survival was only (9 +/- 3)% in the Ad-EF1alpha-CD-CMV-TK group, but (37 +/- 3)% in the Ad-CMV-TK and (46 +/- 4)% in the Ad-EF1alpha-CD groups (P &lt; 0.05). Flow cytometry analysis indicated that the killing mechanisms of the groups were different. Necrosis and apoptosis were involved in the mechanism of the double gene group. Based on the DNA stainability with Hoechst 33342, the apoptotic rates of VSMCs in the Ad-EF1alpha-CD-CMV-TK [(11.0 +/- 2.1)%] and Ad-CMV-TK [(12.0 +/- 2.2)%] groups were higher than those in Ad-CMV-LacZ [(1.2 +/- 0.11)%] and Ad-EF1alpha-CD [(5.0 +/- 1.8)%] groups (P &lt; 0.05, respectively). DNA smear could be observed in both Ad-CMV-TK and Ad-EF1alpha-CD-CMV-TK groups after administration of prodrugs.&lt;/p&gt;&lt;p&gt;&lt;b&gt;CONCLUSIONS&lt;/b&gt;The killing effect on rat VSMCs mediated by adenoviral CD/TK double suicide genes is superior to that of single suicide gene. The killing mechanism of recombinant adenovirus coexpressing CD/TK double suicide genes is mainly through cytotoxic effect and apoptosis.&lt;/p&gt;<p /> <p>Language: en</p>
LA  - en
SN  - 0366-6999
UR  - http://dx.doi.org/
ID  - ref1
ER  -