Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide.
Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm in the murine keratinocyte cell line BALB/MK, induces death in these cells. Cell death is caused by a factor called DOKEB (death factor obtained from keratinocytes expressing bsrm), which is released before the cells' death. In this report we describe and discuss the purification and characterization of DOKEB. Our results were as follows. (i) The 5-day-old medium from the modified BALB/MK cells with LBmSN was used for purification and characterization by filtration and chromatography: DOKEB was a stable and highly hydrophilic compound, with a molecular mass less than that of 1 amino acid. (ii) The conditioned medium containing DOKEB was reactive against thiobarbituric acid and dichlorofluorescein diacetate. (iii) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore, our conclusion is that the BALB/MK cells expressing bsrm produce a large amount of hydrogen peroxide, which catalyzes the process of apoptosis of those cells.
Takeshita
Daniela
D
Department of Biophysics, UNIFESP, São Paulo-SP, Brazil.
Bento
Fernanda Mara
FM
Chammas
Roger
R
Belizário
José Ernesto
JE
Carmona
Adriana Karaoglanovic
AK
Konno
Katsuhiro
K
Lopes de Melo
Robson
R
Molina
Gustavo
G
Lisboa
Bianca Cristina Garcia
BC
Han
Sang Won
SW
D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000302:isolation & purification / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D051017:Apoptosis Regulatory Proteins; D005819:Genetic Markers; D006861:Hydrogen Peroxide; D000619:Aminohydrolases; C019795:blasticidin S deaminase