[In vitro experimental study of killing Hep-2 cells of laryngeal cancer with suicide fusion gene CDglyTK].
OBJECTIVE
To study the killing effect of suicide gene CDglyTK combined with GCV or 5-FC on the human laryngeal carcinoma Hep-2 cell line in vitro.


METHODS
Constructed plasmid pcDNA3.1 (-) CMV. CDglyTK was verified by enzyme digestion of Xho I /Hind III and automatic sequence analysis, then it was introduced into Hep-2 cells by electroporation to yield cells expressing CDglyTK stably after selecting with G418(400 ng/L) for 14 da. The expression of CDglyTK mRNA in transfected Hep-2 cells was tested by RT-PCR. Compared with Hep-2 cells transferred with pcDNA3.1(-), in vitro chemosensitivity of CDglyTK-expressing Hep-2 cells to 5-FC, GCV or 5-FC + GCV was detected by MTT assay.


RESULTS
The recombinant plasmid contained full-length coding region sequence of CD and TK gene. A anticipated 707 bp fragment was amplified from total RNA of CDglyTK-expressing Hep-2 cells by RT-PCR and a fusion protein of 59 000 was detected in cell extract from transfected Hep-2 cells. In vitro study growth of CDglyTK-positive Hep-2 cells were inhibited by 5-FC, GCV or 5-FC + GCV respectively, and the antitumour effect of 5-FC + GCV is superior to 5-FC or GCV.


CONCLUSIONS
CDglyTK may be a candidate for treating human laryngeal cancer.
Tang
Yaoyun
Y
Department of Otolaryngology, Hospital of Xiangya, Central South University, Changsha 410008, China.
Xie
Changning
C
Liu
Jianping
J
Zhao
Suping
S
Tian
Yongquan
Y
Xiao
Jianyun
J
D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000235:genetics; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics