NF-kappaB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: difficulties in identification of dead cells in such cultures.
BACKGROUND
Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI. While studying cytostatic effects of the anti-inflammatory sesquiterpene parthenolide (PRT), we have noticed that, concurrent with apoptosis, the cells were dying by necrosis in the same cultures. Furthermore, because apoptosis was atypical, reflected by rapid loss of plasma membrane integrity, it was difficult to distinguish apoptotic from necrotic cells based on this feature.


METHODS
Three methods were used to distinguish apoptosis from necrosis: (a) HL-60 cells treated with PRT were subjected to analysis of caspases activation using antibody that detects activated (cleaved) caspase-3, (b) apoptotic cells were identified by binding of fluorochrome-labeled inhibitor of caspases FAM-VAD-FMK combined with the PI exclusion assay, and (c) cellular DNA and RNA were differentially stained with acridine orange (AO).


RESULTS
Apoptotic cells were characterized by (a) caspase-3 activation detected immunocytochemically and (b) binding of FAM-VAD-FMK followed by or concurrent with (c) loss of ability to exclude PI, (d) deficit in DNA content, and (e) relatively little changed RNA content. Necrotic cells showed (a) no evidence of caspase-3 activation, (b) no binding of FAM-VAD-FMK, (c) inability to exclude PI, (d) rapid loss of RNA, and (e) unchanged DNA content.


CONCLUSIONS
Identification of apoptotic cells versus necrotic cells was possible either based on the evidence of caspase-3 activation, by labeling with FAM-VAD-FMK combined with PI or by differential staining of cellular DNA and RNA with AO. The data indicate that plasma membrane appears to be one of the targets of PRT, because its integrity is lost very early during cell death, which is reflected by atypical apoptosis and by primary necrosis (lysis of the membrane).
Pozarowski
Piotr
P
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Halicka
Dorota H
DH
Darzynkiewicz
Zbigniew
Z
D000165:Acridine Orange / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009336:Necrosis; D011419:Propidium / Q000494:pharmacology; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012717:Sesquiterpenes / Q000494:pharmacology*; D013997:Time Factors
D000893:Anti-Inflammatory Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D004396:Coloring Agents; D016328:NF-kappa B; D012717:Sesquiterpenes; C002669:parthenolide; D011419:Propidium; D012313:RNA; D004247:DNA; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000165:Acridine Orange