Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation.
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)-alpha or 1 - 40 microg 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-alpha 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.
Solito
E
E
Imperial College School of Medicine, Charing Cross Campus, London, UK.
de Coupade
C
C
Canaider
S
S
Goulding
N J
NJ
Perretti
M
M
D017305:Annexin A1 / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology*; D016718:Arachidonic Acid / Q000378:metabolism; D001562:Benzimidazoles; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D009000:Monocytes / Q000201:enzymology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020298:U937 Cells
D017305:Annexin A1; D001562:Benzimidazoles; D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D016650:Fluorescein-5-isothiocyanate; C017807:bisbenzimide ethoxide trihydrochloride; D002118:Calcium