Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide
Takeshita D.1,7; Bento, F. M.1; Chammas, R.2; Belizário, J. E.3; Carmona A. K.1; Ulbrich, A. G.4, 5; Amarante-Mendes, G. P.4, 5; Konno, K. 6; Han, S. W.1,7.
1 Department of Biophysics, UNIFESP, São Paulo-SP, Brazil; 2 Department of Radiology, FM-USP, São Paulo-SP, Brazil; 3 Department of Pharmacology, ICB-USP, São Paulo-SP, Brazil; 4 Department of Immunology, ICB-USP, São Paulo-SP, Brazil; 5 Institute for Immunology Research, Millenium Institute, São Paulo-SP, Brazil; 6 Butantan Institute, São Paulo-SP, Brazil; 7 Interdisciplinary Center of Gene Therapy, UNIFESP, São Paulo-SP, Brazil.
Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm, in the murine keratinocyte cell line BALB/MK induces death of these cells. The cells die due to a factor, which was named DOKEB (Death factor Obtained from Keratinocytes Expressing Bsrm), released before their death. In this report we describe and discuss the purification and characterization of DOKEB: 1) The five-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 weight lower than one amino acid. 2) The conditioned medium containing DOKEB was reactive against TBA (thiobarbituric acid) and DCFH-DA (dichlorofluorescein diacetate). 3) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore our conclusion is that the BALB/MK cells expressing bsrm suffer oxidative stress and produce an elevated amount of hydrogen peroxide, which catalizes the process of apoptosis of those cells.
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