Cell-surface exposure of phosphatidylserine correlates with the stage of fludarabine-induced apoptosis in chronic lymphocytic leukemia and expressionof apoptosis-regulating genes

Background: Programmed cell death (PCD) is characterized by a sequence of t ightly regulated events that result in the activation of caspases and in in ternucleosomal DNA cleavage. Late apoptotic events such as DNA-strand break s call be assayed by in situ end labeling (ISEL) and DNA measurement (sub G I) using flow cytometry. Phosphatidylserine (PS) redistribution from the: i nner plasma membrane leaflet to the outer Leaflet, an early event in PCD, c an be detected by annexin V (AxV) binding to PS. AxV-fluorescein isothiocya nate (FITC) fluorescence intensity is variable and characterizes different cell populations, denoted here as AxV-negative (AxV(neg)), AxV-low-positive (AxV(lo)), and AxV-high-positive (AxV(hi)). Methods: We investigate the correlation of three methods (ISEL, sub G1 DNA content, and AxV assay) for detecting apoptosis with focus on differences b etween populations with different levels of PS. We also examined the expres sion of PCD-regulating Bcl-2 family members in these cell populations by re verse transcription-polymerase chain reaction (RT-PCR). Chronic lymphocytic leukemia (CLL) cells exposed to fludarabine (FAMP) mere used as an ill vit ro model. Cells with different PS/AxV levels were separated using fluoresce nce-activated cell sorting (FACS). Results: Only purified AxV(hi) cells bad high positivity in the ISEL and su b G1 assays (94 +/- 0.6%, 88.6 +/- 6.6%, and 98.6 +/- 0.6%, respectively) i ndicating that late apoptotic cells are detected equally by all three metho ds. In the AxV(lo) population, ISEL was positive in 21% +/- 13% and DNA sub G1 in 20% +/- 6.6% of cells, suggesting that AxV identifies early apoptoti c cells better than the other assays. Anti-apoptotic Bcl-2 and Bcl-X-L, wer e upregulated by FAMP when cells entered apoptosis (hxV(lo)), as was pro-ap optotic Bcl-X-S, which was undetectable in nonapoptotic AxV(neg) cells. Pro -apoptotic Bar was only expressed in AxV(neg) and AxV(lo) cells. Late apopt otic AxV(hi) cells did not express Bcl-X-S or Bax. Results: (1) AxV staining is more sensitive than sub G1 or ISEL in detectin g early apoptotic cells; (2) only late apoptotic cells are equally detected by all assays; (3) AxV is a valuable tool in the detection and isolation o f apoptotic cells at different stages of PCD; and (4) pro-apoptotic Bcl-X-S and Bax are expressed at early, not late, stages of apoptosis. Cytometry 4 0:19-25: 2000, (C) 2000 Wiley-Liss, Inc.