Life span shortening of normal fibroblasts by overexpression of BCL-2: a result of potent increase in cell death.
It is well known that BCL-2 protects against cell death by both apoptosis and necrosis. The culture of bcl-2-transfected normal fibroblasts showed a shorter life span by about 12 population doubling levels compared to that of vector transfectants (64 vs 76 population doubling levels, respectively). An MTT assay revealed that BCL-2-overexpressing cells (HCA2/bcl-2) showed more severe growth suppression due to hydrogen peroxide or doxorubicin treatment than vector control cells (HCA2/vector). We observed a significant number of dead cells in the HCA2/bcl-2 culture, but not in the HCA2/vector culture. Other BCL-2 family proteins with both antiapoptotic and proapoptotic activity and other apoptosis-related factors were maintained at similar levels, indicating that overexpression of BCL-2 is the major reason that normal fibroblasts are sensitized to cell death. A broad caspase inhibitor (z-Val-Ala-Asp-fmk) and inhibitors of specific caspases (acetyl-Asp-Glu-Val-Asp-CHO, acetyl-Ile-Glu-Thr-Asp-CHO, and acetyl-Leu-Glu-His-Asp-CHO) suppressed cell death of HCA2/bcl-2 effectively, suggesting involvement of caspase 3-, 8-, and 9-dependent pathways in cell death and that the form of death is apoptosis. Unexpectedly, involvement of active MEK in cell death was shown by the use of its inhibitor, suggesting that crosstalk between BCL-2 and the MAP kinase cascade regulates death as well as life span.
Kumazaki
Tsutomu
T
Department of Translational Cancer Research, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553, Japan. kumazaki@hiroshima-u.ac.jp
Sasaki
Masao
M
Nishiyama
Masahiko
M
Teranishi
Yasuhiro
Y
Sumida
Hiroshi
H
Eboshida
Akira
A
Mitsui
Youji
Y
D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002455:Cell Division; D002460:Cell Line; D016922:Cellular Senescence; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D020169:Caspases