Two isoforms of sphingosine kinase (SphK) catalyze the formation of sphingosine-1-phosphate (S1P). Whereas, SphK1 stimulates cellgrowth and survival, it was found that when overexpressed in mouse NIH 3T3 fibroblasts SphK2 enhances caspase-dependent apoptosis inresponse to serum deprivation, independently of S1P receptors. Sequence analysis revealed that SphK2 contains a 9 amino acid motifsimilar to that present in BH3-only proteins. Studies showed that the BH3-only domain and catalytic activity contribute to the apoptoticeffects of overexpressed SphK2. Further studies in human carcinoma cells showed that overexpression of SphK2 increased the expressionof the cyclin dependent kinase (cdk) inhibitor p21, but interestingly had no effect on p53 or its phosphorylation. Correspondingly,downregulation of endogenous SphK2 with small interfering RNA (siRNA) targeted to unique mRNA sequences decreased basal anddoxorubicin-induced expression of p21 without affecting p53. In addition, downregulation of SphK2 decreased G2/M arrest in response todoxorubicin. Surprisingly however, siSphK2 markedly enhanced apoptosis induced by doxorubicin in MCF7 cells. This result raises thequestion of how overexpression of SphK2 decreases cell growth and enhances apoptosis while its downregulation sensitizes cells toapoptosis. A partial answer may come from the possibility that when SphK2 is overexpressed it does not always have the same subcellulardistribution as the endogenous protein. It may also be possible that proteolysis of overexpressed SphK2 might induce apoptosis due toliberation of its BH3 peptide domain, which does not occur at the levels at which endogenous SphK2 is expressed. Collectively, theseresults demonstrate that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest thatSphK2 expression may influence the balance between cytostasis and apoptosis.