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Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase‐3 activation
Author(s) -
Solito Egle,
De Coupade Catherine,
Canaider Silvia,
Goulding Nicolas J,
Perretti Mauro
Publication year - 2001
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0704054
Subject(s) - transfection , microbiology and biotechnology , annexin , apoptosis , biology , clone (java method) , cell culture , phosphatidylserine , programmed cell death , flow cytometry , biochemistry , gene , phospholipid , genetics , membrane
Transfection of the pre‐monomyelocytic U937 cell line with a plasmid coding for full‐length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over‐expression of the N‐terminal and the first domain of the protein (144 amino acids, clone ANX1‐S), which does not contain the Ca 2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml −1 tumour necrosis factor (TNF)‐α or 1 – 40 μg ml −1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1‐AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF‐α increased ANX1 mRNA and protein expression in a time‐dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1‐S showed higher constitutive (more than 2 fold) and activated caspase‐3 activity, associated with higher phospholipase A 2 (PLA 2 ) activity (in the region of +50 – 100%), whereas expression of cytosolic PLA 2 Bax and Bcl‐2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo‐monocytic lineage.British Journal of Pharmacology (2001) 133 , 217–228; doi: 10.1038/sj.bjp.0704054