Phosphatidylserine efflux and intercellular fusion in a BeWo model of human villous cytotrophoblast.
Phosphatidylserine (PS) efflux characterizes cytotrophoblast apoptosis and differentiation. To evaluate whether PS externalization and intercellular fusion were secondary to apoptosis, BeWo cells were induced to differentiate by forskolin or undergo apoptosis by staurosporine. PS externalization was measured by FITC-annexin V binding, and intercellular fusion was quantified by counting nuclei in syncytial cells. During forskolin treatment, vanadate decreased PS efflux by 78.0 per cent from 68.0 [5.3] (mean [SD]) to 15.0 [8.8] Lum (x10(3)) (P<0.001), whereas Z-VAD-fmk had no effect (66.5 [7.3]). Vanadate decreased intercellular fusion from 78.1 per cent [4.1] fusion in uninhibited cultures to 23.4 per cent [2.5], compared with 10.0 per cent [1.7] in media alone. Z-VAD-fmk did not affect fusion (80.4 per cent [6.8]). Staurosporine induced PS efflux was not affected by vanadate (69.6 [5.5] Lum x10(3)), but was inhibited 87.8 per cent by Z-VAD-fmk; from 71.5 [6.2] to 8.7 [3.6] Lum (x10(3)) (P<0.001). Apoptosis was measured by the TUNEL and COMET assays, lamin B fragmentation, activation of procaspase 3, mitochondrial membrane potential, and release of mitochondrial cytochrome c and apoptosis inducing factor. There was no indication of apoptosis associated with differentiation. Thus, PS efflux and intercellular fusion occurred through a vanadate-sensitive mechanism that was independent of apoptosis.
Das
M
M
Department of Obstetrics and Gynecology, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Xu
B
B
Lin
L
L
Chakrabarti
S
S
Shivaswamy
V
V
Rote
N S
NS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051033:Apoptosis Inducing Factor; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002459:Cell Fusion; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000037:antagonists & inhibitors / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D053938:DNA Fragmentation / Q000187:drug effects; D005420:Flavoproteins / Q000378:metabolism; D006801:Humans; D034921:Lamin Type B / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D010718:Phosphatidylserines / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014327:Trophoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D014638:Vanadates / Q000494:pharmacology
C494829:AIFM1 protein, human; D000590:Amino Acid Chloromethyl Ketones; D051033:Apoptosis Inducing Factor; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D005420:Flavoproteins; D034921:Lamin Type B; D008563:Membrane Lipids; D008565:Membrane Proteins; D010718:Phosphatidylserines; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005576:Colforsin; D014638:Vanadates; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine