Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87.
OBJECTIVE
To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells.


METHODS
The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction.


RESULTS
The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium.


CONCLUSIONS
With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.
Wei
X C
XC
Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xue Yuan Road, Haidian District, Beijing 100083, China. xinjuan@sun.bjmu.edu.cn
Wang
X J
XJ
Chen
K
K
Zhang
L
L
Liang
Y
Y
Lin
X L
XL
D016475:3T3 Cells; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D004318:Doxycycline / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D012190:Retroviridae / Q000235:genetics; D013274:Stomach Neoplasms; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D000900:Anti-Bacterial Agents; D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D004318:Doxycycline