Abstract
Aim:
To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells.
Methods:
Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨm) was measured using DiOC6(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays.
Results:
Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨm) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression.
Conclusion:
Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No 30870128), the Ministry of Science and Technology of China (No 2006CB910103 and 2007CB914800), and the 111 Project (No B08011). The authors would like to thank Drs Keith BURRIDGE and Quan CHEN for providing reagents and helpful discussions.
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Supplementary Figure 1
J.Vav1 cells were susceptible to serum starvation and oxidation induced apoptosis. (DOC 490 kb)
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Yin, J., Wan, Yj., Li, Sy. et al. The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells. Acta Pharmacol Sin 32, 99–107 (2011). https://doi.org/10.1038/aps.2010.185
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DOI: https://doi.org/10.1038/aps.2010.185
