Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase‐3 activation

• Published in: British journal of pharmacology 2001-05, Vol.133 (2), p.217-228
• Main Authors: Solito, Egle, De Coupade, Catherine, Canaider, Silvia, Goulding, Nicolas J, Perretti, Mauro
• Format: Article
• Language: English
• Subjects: Ageing, cell death; Annexin A1 - genetics; apoptosis; Apoptosis - genetics; Apoptosis - physiology; Arachidonic Acid - metabolism; Benzimidazoles; Binding Sites - genetics; Biological and medical sciences; Calcium - metabolism; Caspase 3; Caspases - physiology; Cell Adhesion Molecules - metabolism; Cell Cycle - physiology; Cell physiology; Enzyme Activation - physiology; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Human; Humans; lipocortin; Molecular and cellular biology; monocytes; Monocytes - enzymology; Monocytes - metabolism; Mutagenesis, Site-Directed; Plasmids - genetics; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Necrosis Factor-alpha - pharmacology; U937 Cells
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0704054

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 Ca2+ 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 A2 (PLA2) activity (in the region of +50 – 100%), whereas expression of cytosolic PLA2 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