Experimental Study On The Effect Of Gene Apoptin Be Introduced Into And Expressed In Human Colonic Carcinoma Cells

Objective To construct a doxicyclin-regulated recombinant retroviral vector carrying gene apoptin and investigate the effect of gene apoptin expression on Lovo, a human colonic carcinoma cell line in the inducement of doxicyclin. Methods The gene apoptin was cloned into the retroviral vector pRevTRE to obtain the recombinant vector pRevTRE-VP3. The control vector plasmid pRevTet-on and the response vector plasmid pRevTRE-VP3 were packaged with PT67 cell, respectively, and the infectious, replication-incompetent retrovirus were produced. Two retrovirus were, in turn, used to infect Lovo cells. one clone of Lovo/on with highest inducibility was singled out. The supernate of PT67/VP3 was use to infect cell Lovo/on. After antibiotic selection, the double-stable, inducible cell line Lovo/on-VP3 was established. The expression of gene apoptin in Lovo/on-VP3 cells was induced by adding doxicyclin to culture medium. The expression of apoptin and the rate of apoptosis in Lovo/on-VP3 cells was assayed with PCR, RT-PCR, fluorescent staining and flow cytometer (FCM). Results pRevTRE-VP3 was identified by restrictive enzyme digestion and it's sequence was assayed. The highest viral titers after packaged by PT67 were : PT67/on-4-3×10~5cfu/ml and PT67/VP3-2-2×10~5cfu/ml. Two PT67-packaged virus were used to infect cell Lovo, in turn. A double-stable, inducible cell line Lovo/on-VP3-5 that expresses apoptin in the control of doxicyclin was constructed. The integration and expression of gene apoptin in Lovo/on-VP3 was confirmed by PCR and RT-PCR. The result of FCM show that the apoptosis rate in the Lovo/on-VP3 cells incubated in medium with 2mg/l doxicyclin was significantly higher than that incubated in medium without Dox at the same time point. Also, the rate of apoptosis was increased with time to induce, in accord with the result of RT-PCR.Conclusion 1. The gene apoptin can be successfully integrated into genome of Lovo, a human colonic carcinoma cell line with RevTet-on system.2. The expression of gene apoptin can be effectively induced by doxicycline.3. The expression of gene apoptin can induce apoptosis of Lovo cells.