Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.
OBJECTIVE
To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated.


METHODS
Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test.


RESULTS
The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P<0.05, P<0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was >15000 and 214.5+/-31.3 micromol.L(-1), P<0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent.


CONCLUSIONS
The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.
Shen
Li-Zong
LZ
Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, Jiangsu Province, China.
Wu
Wen-Xi
WX
Xu
De-Hua
DH
Zheng
Zhong-Cheng
ZC
Liu
Xin-Yuan
XY
Ding
Qiang
Q
Hua
Yi-Bing
YB
Yao
Kun
K
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000963:Antimetabolites / Q000627:therapeutic use; D024201:Bystander Effect; D002272:Carcinoembryonic Antigen / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015179:Colorectal Neoplasms / Q000235:genetics / Q000503:physiopathology / Q000628:therapy*; D043525:Cytosine Deaminase; D005437:Flucytosine / Q000627:therapeutic use; D015316:Genetic Therapy; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009700:Nucleoside Deaminases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured
D000963:Antimetabolites; D002272:Carcinoembryonic Antigen; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase