U937 variant cells as a model of apoptosis without cell disintegration

Abstract

Abbreviations used: ATCC -American Type Culture Collection; CHX -cycloheximide; DCF -2,7-dichlorofluorescin; DHCF-DA -2',7'-dihydrodichlorofluorescein diacetate; DHE -dihydroethidium; MMP -mitochondrial membrane potential; · O¯2 -superoxide anion; PS -phosphatidylserine; TMRM -tramethylrhodamine methyl ester; TNFα -tumor necrosis factor; U937 V -a variant of the U937 cell line; VP-16 -etoposide Abstract: The variant cell line U937 V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937 V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937 V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937 V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937 V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937 V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.

Defective apoptosis of U937 cells induced by benzyl isothiocyanate (BITC)

“…Negligible cell disassembly, but noticeable granularity of the dying cells were observed. These morphological markers were different in comparison to leukemia cells treated with other death inducers (Stasilojc et al, 2013). Similar morphological changes as BITC-treated U937 cells were observed for Raji and Namalwa cell lines.…”

“…BITC was unable to induce morphological markers of apoptosis in dying U937 cells, which remains in discordance with previous reports that suggests that BITC induces this type of cell death (Stasilojc et al, 2013;Wu et al, 2011). Therefore, we decided to analyze other markers of apoptosis as well.…”

“…Few reports describe cell death of human or murine leukemia cells (Jurkat, HL-60, WEHI-3) treated with BITC (Antony et al, 2012;Coleman et al, 2001;Lee et al, 2009;Zhou et al, 2013). In the study presented here we chose U937 cells as an acknowledged model for a standard caspase-dependent apoptosis (Stasilojc et al, 2013). Our results remain in accordance with previous reports of BITC cytotoxic effects on leukemia cells (Zhang et al, 2003), but indicate that the observed morphological and biochemical markers are not typical for standard apoptotic cell death (Fadok et al, 1992).…”

“…cytes. They were used in studies on cell death (Stasilojc et al, 2013), novel anticancer drugs' evaluation, including ITCs action (Zhou et al, 2013), as well as an in vitro assay to identify contact sensitizers (Python et al, 2007). Apoptosis is characterized by contraction and shrinkage of cytoplasmic organelles, as well as nuclear chromatin condensation (Wickman et al, 2013;Zhou et al, 2013).…”

Synthesis, QSAR studies, and metabolic stability of novel 2‐alkylthio‐4‐chloro‐N‐(5‐oxo‐4,5‐dihydro‐1,2,4‐triazin‐3‐yl)benzenesulfonamide derivatives as potential anticancer and apoptosis‐inducing agents

“…Analysis was performed using FITC‐conjugated annexin‐V (Annexin V‐FITC Apoptosis Kit I, BD Biosciences) according to the manufacturer's instructions (BD Biosciences) as described previously . HeLa cells were coincubated for 24 hr with 33 and 34 .…”