Open AccessEffects of caspase inhibition on camptothecin‐induced apoptosis of HL‐60 cells
Author(s) -
King Malcolm A.,
RadicchiMastroianni Monica A.
Publication year - 2002
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.10141
Subject(s) - propidium iodide , camptothecin , apoptosis , phosphatidylserine , flow cytometry , microbiology and biotechnology , annexin , biology , cell cycle , cell , fluorescein isothiocyanate , chemistry , biophysics , programmed cell death , membrane , biochemistry , fluorescence , phospholipid , physics , quantum mechanics
Background During camptothecin (CAM)‐induced apoptosis of HL‐60 cells, the external exposure of phosphatidylserine (PS) can either precede or follow DNA cleavage. The evidence suggests that cells in S‐phase when CAM is added undergo rapid DNA, nuclear, and cellular disintegration before exposing PS on the outside of the plasma membrane, whereas cells moving from G1 into S‐phase after CAM is added expose PS before they manifest the other phenomena. This study describes further investigations using the broad spectrum caspase inhibitor Z‐VAD‐FMK. The cells were cultured for a period long enough to ascertain whether a particular phenomenon was only delayed or was blocked completely. Methods Changes in cell light scatter, binding of annexin V‐fluorescein isothiocyanate (FITC) to PS, uptake of propidium iodide (PI) as a measure of plasma membrane integrity, and DNA content after membrane fixation/permeabilization were monitored by flow cytometry during 24‐h cultures. Fluorescence microscopy was used to examine cell morphology. Results Caspase inhibition blocked DNA cleavage, breakdown of the nuclear membrane, and formation of apoptotic bodies. It also revealed the existence of a CAM‐activated early S‐phase checkpoint. Cells arrested in early S‐phase preceded the appearance of PS‐positive cells. Caspase inhibition delayed both PS exposure and loss of plasma membrane integrity but did not prevent either. Conclusions The results support the hypothesis that the sequence of apoptotic phenomena in an individual CAM‐treated HL‐60 cell depends on the stage of proliferation of that cell when it encounters the CAM. They are also consistent with the hypothesis that caspases are not required for PS exposure or the loss of plasma membrane integrity, but they are involved indirectly in promoting these phenomena. Cytometry 49:28–35, 2002. © 2002 Wiley‐Liss, Inc.