Analysis of bax protein in sphingosine-induced apoptosis in the human leukemic 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 sphingosine in bcl-2-overexpressing cells compared with cells that do not express the bcl-2 gene. The human erythroleukemic cell line TF1, which lacks bcl-2 expression, was easily induced to undergo apoptotic cell death by a variety of stimuli, including depletion of granulocyte-macrophage colony-stimulating factor (GM-CSF) or exposure to methylmethane sulfonate (MMS) (100 microg/mL), ultraviolet light (15 J/m2), X-ray irradiation (20 Gy), or sphingosine, a sphingolipid breakdown product (5 microM). 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 ceramide) induced apoptosis in TF1-mock and TF1-bcl2 cells. Sphingosine-induced apoptosis could not be inhibited by fumonisin B1, which can prevent conversion of sphingosine to ceramide, suggesting that sphingosine itself, not ceramide, possesses apoptosis-inducing capability. Western blotting, which revealed a 21-kDa bax protein in untreated cells, revealed the presence of an additional 18-kDa protein in GM-CSF-depleted and MMS- or sphingosine-treated TF1-mock cells. In TF1-bcl2 cells, this protein was not detected after GM-CSF depletion or MMS treatment, but was observed after sphingosine treatment. Immunoprecipitation with anti-bcl2 antibody, followed by immunoblotting with anti-bax antibody, showed that both the 21-kDa bax protein and the 18-kDa protein heterodimerized with bcl-2 protein. These results suggest that sphingosine is a unique reagent for apoptosis and that it can overcome bcl-2 gene expression. Furthermore, induction of 18-kDa bax-related protein may play an important role in apoptosis. Sphingosine, but not ceramide, may prove applicable as a reagent for future cytotoxic drugs used to treat intractable tumors overexpressing bcl-2.
Isogai
C
C
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Murate
T
T
Tamiya-Koizumi
K
K
Yoshida
S
S
Ito
T
T
Nagai
H
H
Kinoshita
T
T
Kagami
Y
Y
Hotta
T
T
Hamaguchi
M
M
Saito
H
H
D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002264:Carboxylic Acids / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004351:Drug Resistance / Q000235:genetics; D037341:Fumonisins; D006801:Humans; D009183:Mycotoxins / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013110:Sphingosine / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D002264:Carboxylic Acids; D018076:DNA, Complementary; D037341:Fumonisins; D009183:Mycotoxins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C056933:fumonisin B1; D004247:DNA; D013110:Sphingosine