Acute changes in U937 nuclear Ca2+ preceding type 1 "apoptotic" programmed cell death due to MK 886.
BACKGROUND
MK 886, a 5-lipoxygenase inhibitor, induces a type 1 "apoptotic" form of programmed cell death in Bcl-2-positive U937 monoblastoid cells. In Ca2+-depleted, nonpermeabilized U937 cells studied with MK 886 in a Ca2+-free medium, an acute increase in Ca2+ occured within 10 to 20 seconds, detected with fura-2 measured with a spectrofluorimeter.


RESULTS
The increased fluorescence was nuclear in location, as judged by confocal microscopy. The antioxidant, N-acetyl-L-cysteine, three agents that inhibit mitochondrialfunction at identified sites, antimycin A, atractyloside and cyclosporin A, the L/N-channel inhibitor, loperamide and BAPTA, an intracellular Ca+ chelator preloaded into cells each reduced the extent or prevented the acute MK 886-induced rise in Ca2+, as determined by radiometric detection. Rhodamine-2, a more selective mitochondrial Ca2+ probe, provided no evidence for nuclear Ca2+ originating from that extra-nuclear site or from the endoplasmic reticulum. With 2', 7'-dichloro-dihydrofluorescein-labelled cells to detect reactive oxygen species, MK 886 increased the initial fluorescent signal from a number of intracellular, largely extra-nuclear sites, including mitochondria. Two chemicals that inhibit the function of Bcl-2, HA14-1 and 2-methyl-antimycin A3, reduced the Ca2+ response to MK 886, if pre-incubated with the Bcl-2-positive U937 cells at 37 degrees C for several hours. MK 886 was previously shown to induce reactive oxygen species and a fall in mitochondrial membrane potential in both Bcl-2-positive U937 and in Bcl-2-negative PC-3 prostate and panc-1 pancreatic cancer cells. The latter solid tumor cells undergo an atypical "type 2" PCD without an acute rise in nuclear Ca2+.


CONCLUSIONS
These results are consistent with an MK 886-induced increase of reactive oxygen species from intra-cellular sites including mitochondria which release Ca2+ located primarily at or near nuclei. These events may involve Bcl-2 participating in some form of Ca2+ channel and nuclear Ca2+ binding proteins undergoing conformational changes due to reactive oxygen species. Reasons for the different PCD responses in Bcl-2 positive lympho-hematopoietic compared to Bcl-2-negative solid cancer cell lines, respectively with and without the induced nuclear Ca2+ signal, remain to be defined.
Anderson
K M
KM
Hektoen Institute, Department of Biochemistry, Rush University Medical Center, University of Illinois and West Side V.A. Chicago, IL 60612, USA. marander427@MSN.com
Rubenstein
M
M
Alrefai
W A
WA
Dudeja
P
P
Tsui
P
P
Harris
J E
JE
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism / Q000494:pharmacology; D006575:Heterocyclic Compounds, 3-Ring; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020298:U937 Cells; D002722:Chlorobenzenes
D002118:Calcium; D004533:Egtazic Acid; D005456:Fluorescent Dyes; D016257:Fura-2; D006575:Heterocyclic Compounds, 3-Ring; D007211:Indoles; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C060893:MK-886; D002722:Chlorobenzenes; C068483:rhod-2; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid