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.
Guan
Jian
J
Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. chenj@csc.pumch.ac.cn
Chen
Jie
J
Zhao
He
H
D017209:Apoptosis / Q000235:genetics*; D015153:Blotting, Western; D002455:Cell Division / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D016373:DNA, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D013752:Tetracycline