Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macro phage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dep endent manner as revealed by flow cytometry; (2) ConA elicited DNA fragment ation and the cytotoxicity of ConA was suppressed by alpha -D-methylmannosi de which blocks the lectin site of ConA; (3) ConA was able to release cytoc hrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochond ria were incubated with ConA, release of cyto c was observed too. Interesti ngly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC a nd subsequently with mitotracker red (a probe for mitochondria), co-localiz ation of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and re leased cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. O n the other hand, introduction of specific antibody against cyto c into PU5 -1.8 cells suppressed the ConA-mediated cell death. Taken together, our res ults indicate that ConA induced apoptosis in PU5-1.8 cells through mitochon drial clustering and release of cyto c and the release of cyto c was suffic ient to elicit apoptosis in PU5-1.8 cells.