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 (ΔΨ(m)) was measured using DiOC(6)(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 (ΔΨ(m)) 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.
AIM: To investigate a novel function of proto-oncogene Vav1 in the apoptosis of humanleukemia 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 (ΔΨ(m)) was measured using DiOC(6)(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 (ΔΨ(m)) 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.
Authors: David A Hildeman; Yanan Zhu; Thomas C Mitchell; Philippe Bouillet; Andreas Strasser; John Kappler; Philippa Marrack Journal: Immunity Date: 2002-06 Impact factor: 31.745
Authors: Maša Umiċeviċ Mirkov; Jing Cui; Sita H Vermeulen; Eli A Stahl; Erik J M Toonen; Remco R Makkinje; Annette T Lee; Tom W J Huizinga; Renee Allaart; Anne Barton; Xavier Mariette; Corinne Richard Miceli; Lindsey A Criswell; Paul P Tak; Niek de Vries; Saedis Saevarsdottir; Leonid Padyukov; S Louis Bridges; Dirk-Jan van Schaardenburg; Tim L Jansen; Ellen A J Dutmer; Mart A F J van de Laar; Pilar Barrera; Timothy R D J Radstake; Piet L C M van Riel; Hans Scheffer; Barbara Franke; Han G Brunner; Robert M Plenge; Peter K Gregersen; Henk-Jan Guchelaar; Marieke J H Coenen Journal: Ann Rheum Dis Date: 2012-12-11 Impact factor: 19.103