C2-ceramide as a cell death inducer in HC11 mouse mammary epithelial cells.
Ceramide is a lipid mediator in cell proliferation, differentiation, and apoptosis in many cell lines. However, the molecular mechanisms for ceramide have not been clarified in HC11 mouse mammary epithelial cells. Under phase contrast microscope, C2-ceramide-treated cells clearly showed morphological changes, which were characteristic features of apoptosis. Treatment with C2-ceramide at 10 microM specifically resulted in the death of 50% of the cells after 48 h as assessed by MTT assay. To further investigate which genes contribute to cell death in C2-ceramide-treated cells, we used the reverse transcription-polymerase chain reaction to assess mRNA levels for five genes in the Bcl-2 family and five genes in the caspases family. The steady-state mRNA levels of Bax, Bad and Bak were not significantly changed for 48 h of C2-ceramide treatment. The increases of mRNA levels of Bcl-2 and Bcl-w were observed for the first 3 h of C2-ceramide treatment and the last 24 h between 24 and 48 h. We also found that in HC11 cells, C2-ceramide increased mRNA levels of the caspases family from 6 to 24 h. These results suggest that in the HC11 cells, C2-ceramide promote cell death by mediating the induction of caspases and that HC11 mouse mammary epithelial cells paradoxically up-regulate the expression of Bcl-2 and Bcl-w to prevent C2-ceramide-mediated cell death.
Kim
Sung Hak
SH
Department of Animal Science and Technology, School of Agricultural Biotechnology, Seoul National University, Suweon 441-744, South Korea.
Kim
Sung Chan
SC
Kho
Yoon Jung
YJ
Kwak
Sung Wook
SW
Lee
Hong Gu
HG
You
Seung Kwon
SK
Woo
Jung Hee
JH
Choi
Yun Jaie
YJ
D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D045744:Cell Line, Tumor; D002470:Cell Survival; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology*; D004396:Coloring Agents / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology*; D006801:Humans; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000473:pathology*; D051379:Mice; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D002518:Ceramides; D004396:Coloring Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D012313:RNA; C022616:thiazolyl blue