Effects of caspase inhibition on camptothecin-induced apoptosis of HL-60 cells.
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.
King
Malcolm A
MA
Department of Clinical Immunology, Pacific Laboratory Medicine Services, Royal North Shore Hospital, St. Leonards, New South Wales, Australia. making@doh.health.nsw.gov.au
Radicchi-Mastroianni
Monica A
MA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D002166:Camptothecin / Q000494:pharmacology*; D061945:Caspase Inhibitors; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002166:Camptothecin