Effects of tetracycline-controlled antisense bcl-2 expression on the growth and apoptosis of human neuroblastoma cell line SK-N-MC.

OBJECTIVE: To study the effects of tetracycline-controlled antisense bcl-2 expression on the growth and apoptosis of human neuroblastoma cell line SK-N-MC and the related mechanisms. METHODS: The tetracycline-controlled antisense bcl-2 expressing vector PUCCOMB1(CMV)/Asbcl-2 was constructed by inserting a 0.6 kb fragment antisense bcl-2 cDNA sequence into the plasmid PUCCOMB1(CMV). SK-N-MC cells were transfected with PUCCOMB1(CMV)-Asbcl-2 or PUCCOMB1(CMV) by Lipofectamine( trade mark ). The transfectant cells were further studied for growth viability and apoptosis induced by antisense bcl-2 expression controlled by tetracycline. The expression of bcl-x(L)/bcl-x(S) mRNA was examined by RT-PCR and the changes of expression of proteins caspase-3, bcl-2 and PARP were examined by Western-blot. RESULTS: The cell viability of the antisense bcl-2 transfected cells decreased significantly, and the apoptotic cells and DNA ladder could be found earlier in the antisense bcl-2 cells than in the empty vector transfected cells. We also found a decrease of non-activated caspase-3, cleavages of PARP and bcl-2 protein after treatment without fetal bovine serum (FBS) by Western-blot, but there was no change in the expression of bcl-x(L)/bcl-x(S) mRNA examined by RT-PCR during apoptosis. CONCLUSIONS: The results suggested that tetracycline-controlled expression of antisense bcl-2 can effectively inhibit the cell viability of SK-N-MC cells, increase the sensitivity to apoptosis-inducing factor, as well as facilitate cell apoptosis after treatment of culture without FBS. Except bcl-x(L)/bcl-x(S) mRNA expression, activation of caspase-3, cleavage of protein PARP and bcl-2 were all associated with apoptosis.