Acute Changes in U937 Nuclear Ca²⁺ Preceding Type 1 “Apoptotic” Programmed Cell Death due to MK 886

Abstract

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, non-permeabilized 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. Methods and Results: The increased fluorescence was nuclear in location, as judged by confocal microscopy. The antioxidant, N-acetyl-L-cysteine, three agents that inhibit mitochondrial function 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°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⁺. Conclusion: 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.