Influence of culture medium pH on cytotoxicity of CD95L in vitro.
CD95L belongs to the tumor necrosis factor-alpha (TNF-alpha) family, the members of which induce apoptosis by activation of their specific receptors. However, there are a few publications suggesting that two of these factors, TNF-alpha and TNF-beta, are able to reveal cytotoxic effect in pH-dependent manner. Therefore we investigated, whether CD95L may also reveal pH-dependent cytotoxicity. We analyzed influence of CD95L on U937 and K562 human cell lines at pH 5.1 and pH 7.4 using radioactive chromium release and tetrazolium salt (MTT) reduction assays. Expression of CD95 in both cell lines was estimated using RNase Protection Assay and FACS analysis. It has been found that short incubation of cells at pH 5.1 did not visibly affect their viability, as measured after 16 or 20 h. Incubation of U937 with CD95L at pH 7.4 resulted in a dose-dependent cell cytotoxicity. The effect was significantly augmented by incubation of cells with CD95L at pH 5.1. K562 cell line was resistant to CD95L at pH 7.4. This result correlated with the lack of CD95 expression in K562 cells. However, incubation at pH 5.1 resulted in a sensitization of K562 cells to CD95L. Our results suggest that CD95L, similarly to TNF-alpha, is able to reveal its cytotoxic activity in a receptor-independent manner and this activity strongly depends on pH of the environment.
Grzela
T
T
Department of Histology and Embryology, Center of Biostructure Research, Medical University, Warsaw, Poland. tgrzela@ib.amwaw.edu.pl
Lazarczyk
M
M
Ołdak
M
M
Korczak-Kowalska
G
G
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003470:Culture Media / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D020014:K562 Cells; D008562:Membrane Glycoproteins / Q000633:toxicity*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D020298:U937 Cells; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003470:Culture Media; C505533:FADD protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D019014:fas Receptor; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases