[The cloning of human Smac gene and its pro-apoptotic effect on Burkitt's lymphoma cells].
OBJECTIVE
Second mitochondria-derived activator of caspase (Smac) is a recently identified, novel pro-apoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac promotes activation of caspases by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The objective of the study was to examine the pro-apoptotic effect of human Smac gene on Burkitt's lymphoma Raji cells.


METHODS
The full length cDNA of human Smac gene was amplified by reverse transcription-PCR from total RNA of HEK-293 cells. The PCR product was ligated with linearized vector pGEM-T-easy supplied in the TA cloning kit and sequenced. The correct cDNA of full length Smac was subcloned into eukaryocytic expression vector pcDNA3.1/myc-his and transfected into human Burkitt's lymphoma cell line Raji by lipofectamine-mediated transfection. The expression of full length Smac was determined by Western blot. Morphological observation was done with the laser scanning confocal microscope by double staining the Raji cells with Hoechest 33,258 and propidium iodide. Flow cytometry was used to evaluate apoptosis. Relative caspase-3 activity was determined by colorimetric assay.


RESULTS
Recombinant eukaryocytic expression vector pcDNA3.1/Smac, which contained full length Smac, was successfully constructed. After pcDNA 3.1/Smac was transfected into human Burkitt's lymphoma Raji cell line for 24 hours, Raji cells showed apparent apoptosis with a percentage of (43.7 +/- 2.5)%, which was higher than that of non-transfected group and free vector-transfected group (P < 0.05). Compared with non-transfected group (0.136 +/- 0.036) and free vector-transfected group (0.138 +/- 0.026), the relative caspase-3 activity of Raji cells transfected by pcDNA3.1/Smac (0.936 +/- 0.041) was significantly enhanced (P < 0.05).


CONCLUSIONS
Transfection and expression of human Smac gene could significantly induce apoptosis of human Burkitt's lymphoma Raji cells. The mechanism is associated with the increase of caspase-3 activity.
Lu
Chao
C
Department of Pediatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Wu
Sheng-hua
SH
Chen
Ji-qing
JQ
Zhao
Fei
F
Chi
Xia
X
Pan
Xiao-qin
XQ
Fei
Li
L
Guo
Mei
M
Huang
Song-ming
SM
Guo
Xi-rong
XR
Chen
Rong-hua
RH
D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002051:Burkitt Lymphoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D018076:DNA, Complementary; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D053148:Caspase 3