ANALYSIS OF BAX PROTEIN IN SPHINGOSINE-INDUCED APOPTOSIS IN THE HUMANLEUKEMIC-CELL LINE TF1 AND ITS BCL-2 TRANSFECTANTS

Sphingosine, a sphingolipid breakdown product, has been proposed as an apoptosis-inducing agent. In this study, we examined the effect of sp hingosine in bcl-2-overexpressing cells compared with cells that do no t express the bcl-2 gene. The human erythroleukemic cell line TF1, whi ch lacks bcl-2 expression, was easily induced to undergo apoptotic cel l death by a variety of stimuli, including depletion of granulocyte-ma crophage colony-stimulating factor (GM-CSF) or exposure to methylmetha ne sulfonate (MMS) (100 mu g/mL), ultraviolet light (15 J/m(2)), X-ray irradiation (20 Gy), or sphingosine, a sphingolipid breakdown product (5 mu M) In contrast, bcl-2 transfectants of TF1 (TF1-bcl2), which we established, were resistant to most of these treatments but remained sensitive to sphingosine. Neither C2- nor C6-ceramide (short-chain cer amide) induced apoptosis in TF1-mock and TF1-bcl2 cells. Sphingosine-i nduced apoptosis could not be inhibited by fumonisin B1, which can pre vent conversion of sphingosine to ceramide, suggesting that sphingosin e itself, not ceramide, possesses apoptosis-inducing capability. Weste rn blotting, which revealed a 21-kDa bar protein in untreated cells, r evealed the presence of an additional 18-kDa protein in GMCSF-depleted and MMS- or sphingosine-treated TF1-mock cells. In TF1-bcl2 cells, th is protein was not detected after GMCSF depletion or MMS treatment, bu t was observed after sphingosine treatment. Immunoprecipitation with a nti-bcl2 antibody, followed by immunoblotting with anti-bar antibody, showed that both the 21-kDa bar protein and the 18-kDa protein heterod imerized with bcl-2 protein. These results suggest that sphingosine is a unique reagent for apoptosis and that it can overcome bcl-2 gene ex pression. Furthermore, induction of 18-kDa bar-related protein may pla y an important role in apoptosis. Sphingosine, but not ceramide, may p rove applicable as a reagent for future cytotoxic drugs used to treat intractable tumors overexpressing bcl-2.