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[The cloning of human Smac gene and its pro-apoptotic effect on Burkitt's lymphoma cells]
Chao Lu1, Sheng-hua Wu, Ji-qing Chen
1Department of Pediatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
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).
Conclusion:
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.
Insights
Introducing human Smac (second mitochondria-derived activator of caspase) gene into Burkitt
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Death Mechanisms
Background:
- Second mitochondria-derived activator of caspase (Smac) is a pro-apoptotic molecule released during apoptosis.
- Smac neutralizes inhibitor of apoptosis proteins (IAPs), such as XIAP, to promote caspase activation.
- Burkitt's lymphoma is an aggressive non-Hodgkin B-cell lymphoma with a need for novel therapeutic targets.
Purpose of the Study:
- To investigate the pro-apoptotic effects of the human Smac gene on Raji cells, a human Burkitt's lymphoma cell line.
- To determine if Smac expression can induce apoptosis in this cancer cell model.
Main Methods:
- Human Smac cDNA was amplified and cloned into a eukaryotic expression vector (pcDNA3.1/myc-his).
- The expression vector was transfected into Raji cells using lipofectamine.
- Apoptosis was assessed via morphological observation (Hoechst 33,258/propidium iodide staining) and flow cytometry; caspase-3 activity was measured colorimetrically.
Main Results:
- Successful construction and transfection of the pcDNA3.1/Smac vector into Raji cells.
- Transfection with pcDNA3.1/Smac significantly induced apoptosis in Raji cells (43.7% +/- 2.5%) compared to controls (P < 0.05).
- Relative caspase-3 activity was significantly enhanced in Smac-transfected cells (0.936 +/- 0.041) compared to controls (P < 0.05).
Conclusions:
- Expression of the human Smac gene effectively induces apoptosis in human Burkitt's lymphoma Raji cells.
- The pro-apoptotic mechanism involves a significant increase in caspase-3 activity.
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