Increase of ceramide in adriamycin-induced HL-60 cell apoptosis: detection by a novel anti-ceramide antibody.
We recently raised an IgM class of monoclonal antibody (Ab) for ceramide (NHCER-2), and examined its specificity and sensitivity. Enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) showed that NHCER-2 recognized ceramides but not other sphingolipids such as sphingosine, sphinganine, sphingomyelin, sphingosine-1-phosphate, ceramide-1-phosphate, glucosylceramide and cerebroside. In addition, N-hexanoyl, N-octanoyl and N-palmitoylsphingosine were detected by NHCER-2, but N-acetylsphingosine and dihydroceramide were not. Densities of ceramide detected by NHCER-2 were proportional to the amounts of ceramide standard up to 250 ng. When various concentrations of adriamycin (ADR) was added to induce apoptosis, the amounts of ceramide detected by NHCER-2 time- and dose-dependently increased in apoptosis-sensitive HL-60 cells as well as by DGK assay, but not in apoptosis-resistant HL-60/ADR cells. After cell fractionation, ceramide levels judged not only by diacylglycerol kinase (DGK) assay but also by NHCER-2 were shown to increase in the microsomal and the nuclear fraction in apoptosis-sensitive cells, but not in apoptosis-resistant cells. Moreover, absolute amounts of ceramide determined by NHCER-2 were well correlated with those by DGK assay. These results suggest that increase of ceramide in the nuclear fraction as well as in the microsomal fraction may play a role in ADR-induced apoptosis and that a novel anti-ceramide Ab NHCER-2 could be beneficial to investigate changes of ceramide content in the cells.
Kawase
Mamoru
M
Diagnostic Research and Development Department, R&D Division, Nesco Company, Azwell Inc., 2-24-3 Sho, Ibaraki, Osaka 567-0806, Japan.
Watanabe
Mitsumasa
M
Kondo
Tadakazu
T
Yabu
Takeshi
T
Taguchi
Yoshimitsu
Y
Umehara
Hisanori
H
Uchiyama
Takashi
T
Mizuno
Koji
K
Okazaki
Toshiro
T
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D017209:Apoptosis; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002518:Ceramides / Q000032:analysis* / Q000276:immunology; D002855:Chromatography, Thin Layer; D019852:Diacylglycerol Kinase; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay; D018922:HL-60 Cells / Q000187:drug effects*; D006801:Humans; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002518:Ceramides; D004317:Doxorubicin; D019852:Diacylglycerol Kinase