Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

Abstract

Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosisassociated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.

E2F1 downregulation by arsenic trioxide in lung adenocarcinoma

“…Although the induction of cell death by ATO has been investigated extensively in different cancer models, only a few reports have shown ATO-induced PS externalization (37)(38)(39)(40)(41). To our knowledge, this is the first report of PS externalization in an ATO-treated lung cancer cell line (H23).…”

Immuno-analysis of microparticles: probing at the limits of detection

“…Both ligands showed increased labelling on larger MP, as expected, however measuring the median fluorescence intensity relative to arbitrary MP size indicated that this difference is more pronounced with annexin V compared to lactadherin. In the literature it has been shown that annexin V clustering results in its preferential binding to flat membranes and that a threshold of PS must be met for adequate binding, whilst lactadherin has a preference for sharp curvatures and binds PS proportionately to the levels available 11 36 37 38 . Therefore, from our results we propose that the increased labelling intensity of annexin V following TNF activation may be explained by, 1) the increased availability of larger and therefore flatter MP, and 2) discrete changes in PS exposure at the membrane surface which exceed the required threshold.…”

Avenues to molecular imaging of dying cells: Focus on cancer

“…In future studies, lactadherin may be engineered in such a way that only the C2 domain, responsible for PS‐binding, is used for labeling. A fluorescent derivative of the C2 domain has shown the ability to label different cellular pools of PS and apoptotic tumor cells …”

“…A fluorescent derivative of the C2 domain has shown the ability to label different cellular pools of PS 88,92 and apoptotic tumor cells. 208,213,214…”