Withdrawal of 2-mercaptoethanol induces apoptosis in a B-cell line via Fas upregulation.
Mouse lymphoid cell cultures are dependent on reducing agents in their culture medium to allow proliferation and survival of the cells. In the case of the mouse CD5+-pre-B cell line SPGM-1, withdrawal of 2-mercaptoethanol (2-ME) resulted in rapid inhibition of proliferation and subsequent cell death by apoptosis. The pathways leading to cell death by withdrawal of 2-ME or by incubation with ionomycin, a known inducer of apoptosis, were compared. Both kinds of stimulation resulted in apoptosis of the whole population, but cell death occurred with different kinetics. Only apoptosis induced by ionomycin was inhibited by coincubation with the phorbol ester PMA, while apoptosis induced by withdrawal of 2-ME was not. Overexpression of the human bcl-2 proto-oncogene in these cells delayed the death process induced by either method. SPGM-1xbcl-2 cells accumulated in the G0/G1 and G2/M cell cycle phases after removal of 2-ME from the medium, whereas treatment with ionomycin resulted in an arrest only in the G0/G1 transition. Interestingly, both stimuli induced the expression of the Fas receptor, but with different kinetics, while the Fas ligand (FasL) was expressed constitutively in SPGM-1 cells. These data demonstrate that withdrawal of 2-ME and incubation with ionomycin both induce rapid cell death by apoptosis, possibly mediated by an autocrine Fas/FasL loop. Although the initial pathways activated by the two forms of treatment must be different, they converge on a common level controlled by the anti-apoptotic gene product Bcl-2.
Neumann
D
D
Institute for Clinical Molecular Pharmacology, Medical School, Hannover, Germany. biology@dompe.it
Zierke
M
M
Martin
M U
MU
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001402:B-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000473:pathology; D002273:Carcinogens / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D053222:Fas Ligand Protein; D015870:Gene Expression / Q000187:drug effects; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics; D008623:Mercaptoethanol / Q000494:pharmacology*; D051379:Mice; D009336:Necrosis; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019014:fas Receptor / Q000235:genetics*
D002273:Carcinogens; D003470:Culture Media; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007476:Ionophores; C000715470:MAS1 protein, human; D008562:Membrane Glycoproteins; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor; D015759:Ionomycin; D008623:Mercaptoethanol; D013755:Tetradecanoylphorbol Acetate