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

1 Transfection of the pre-monomyelocytic U937 cell line with a plasmid codi ng for full-length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, overexpression 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 fol lowing activation by either 5 ng ml(-1) tumour necrosis factor (TNF)-alpha or 1-40 mug ml(-1) etoposide. This was demonstrated by using the fluorescen t labelled annexin V, cell cycle and nuclear staining analyses. 2 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 th e degree of apoptosis promoted by either stimulant. 3 Treatment of CMV U937 cells with TNF-c( increased ANX1 mRNA and protein e xpression in a time-dependent manner, with maximal increases at 3 and 6 h, respectively. 4 Clone ANX1-S showed higher constitutive (more than 2 fold) and activated caspase-3 activity, associated with higher phospholipase A(2) (PLA(2)) acti vity tin the region of + 50-100%), whereas expression of cytosolic PLA(2) P ax and Bcl-2 were similar in all cell clones, as determined by Western blot ting. 5 In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo-monocytic l ineage.