Bax upregulation is an early event in cisplatin-induced apoptosis in human testicular germ-cell tumor cell line NT2, as quantitated by flow cytometry.
Expression of the apoptosis-associated proteins Bcl-2 and Bax was quantitated by flow cytometry (FCM) in chemosensitive testicular germ-cell tumor NT2 cells, and the results were compared with those obtained by Western blotting. NT2 cells were incubated with cisplatin (3.1 microM for 2 h at 37 degrees C), and 24, 48, and 72 h later were analyzed for induction of apoptosis, and for modulation of the expression of cell death suppressing protein Bcl-2, as well as cell death promoting protein Bax. Apoptosis was quantitated by binding of annexin V conjugated with fluorescein isothiocyanate (FITC) to the cell membrane. Cisplatin-treatment induced apoptosis in NT2 cells. The apoptotic cell population increased in time, and at t = 72 h after drug incubation, about 90% of cells that were present in the cell culture were apoptotic. Subsequently, we determined the expression of the Bcl-2 and Bax proteins by FCM and Western blotting before and after drug treatment. NT2 cells had low constitutive expression levels of Bcl-2 and elevated constitutive expression levels of Bax protein, as determined by both methods. At t = 24 h and 48 h after drug treatment, no changes were observed in the expression of the Bcl-2 protein, as quantitated by FCM and Western blotting. Also, the expression of the Bax protein had not changed, based on Western blotting. However, FCM revealed that in a specific subpopulation of drug-treated NT2 cells, Bax expression was increased. On the basis of forward and perpendicular light-scatter this subpopulation, which consisted of large, early apoptotic, swollen cells with increased internal complexity, was sorted, and showed abundant Bax protein by FCM and Western blotting. Our results demonstrate that the chemosensitivity of NT2 cells is probably due to a low intrinsic threshold for drug-induced apoptosis that is accompanied by overexpression of the death-promoting Bax protein during the early stages of the apoptotic process. We conclude that FCM is superior to Western blotting for the detection of heterogeneous expression of Bax in a given cell population.
Boersma
A W
AW
Department of Medical Oncology, University Hospital Rotterdam, The Netherlands.
Nooter
K
K
Burger
H
H
Kortland
C J
CJ
Stoter
G
G
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002448:Cell Adhesion; D002469:Cell Separation; D002945:Cisplatin / Q000494:pharmacology*; D005434:Flow Cytometry; D018237:Germinoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008297:Male; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013736:Testicular Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D002945:Cisplatin