EXPRESSION OF NEUTRAL SPHINGOMYELINASE IDENTIFIES A DISTINCT POOL OF SPHINGOMYELIN INVOLVED IN APOPTOSIS

The activation of sphingomyelinase and generation of ceramide have bee n implicated as important regulatory pathways in cell growth and apopt osis, Bacterial sphingomyelinase has been used in many cell systems to mimic the activation of endogenous sphingomyelinase. These studies, h owever, have been complicated by the inability of exogenously applied bacterial sphingomyelinase to perform many of the effects of short cha in cell permeable ceramides, indicating that there may be a distinct s ignal transducing pool of sphingomyelin not accessible to exogenous sp hingomyelinase or that endogenous ceramide is not sufficient to induce these changes, We cloned the Bacillus cereus sphingomyelinase gene by polymerase chain reaction and subcloned it into a mammalian expressio n vector under the control of an inducible promoter, Upon stable trans fection and induction of B. cereus sphingomyelinase, there were increa ses in neutral sphingomyelinase activity, cellular ceramide levels, cl eavage of the death substrate poly(ADP-ribosyl)polymerase, and cell de ath, In contrast, exogenously applied B. cereus sphingomyelinase, desp ite causing higher elevations in ceramide levels, was unable to induce poly(ADP-ribosyl)polymerase cleavage or cell death, These results sup port the existence of a signal transducing pool of sphingomyelin that is distinct from the pool accessible to exogenous sphingomyelinase.