Bax upregulation is an early event in cisplatin-induced apoptosis in human testicular germ-cell tumor cell line NT2, as quantitated by flow cytometry

Abstract

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 μM for 2 h at 37°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. Cytometry 27:275–282, 1997. © 1997 Wiley‐Liss, Inc.

Adenovirus‐mediated Gene Transduction of IkB or IkB Plus Bax Gene Drastically Enhances Tumor Necrosis Factor (TNF)‐induced Apoptosis in Human Gliomas

“…[52][53][54] Chemotherapeutic agents also induce cancer cell death through upregulation of Bax. 55) Therefore, our result might be consistent with the report by Wang et al, 56) who had shown that Adv-IκBdN sensitizes chemoresistant tumors to a chemotherapeutic compound, CPT-11. In their report, the inactivation of NF-κB by Adv-IκBdN and the activation of Bax by chemotherapy might have synergistically promoted tumor regression.…”

Relative level of expression of Bax and Bcl-XL determines the cellular fate of apoptosis/necrosis induced by the overexpression of Bax

“…Kitada et al (1996) demonstrated that g-radiation applied to radiosensitive cells upregulated expression of the Bax protein and induced apoptosis. The anti-cancer drugs cisplatin, etoposide, and paclitaxel, exert cytotoxicity by triggering the upregulation of Bax (Boersma et al, 1997), and Baxinduced apoptosis (Chresta et al, 1996;Strobel et al, 1996;Wagener et al, 1996). McCurrach et al (1997) showed that ®broblasts obtained from mice in which the Bax gene had been knocked out, were resistant to chemotherapy.…”

Development of a Human Neuronal Cell Model for Human Immunodeficiency Virus (HIV)-Infected Macrophage-Induced Neurotoxicity: Apoptosis Induced by HIV Type 1 Primary Isolates and Evidence for Involvement of the Bcl-2/Bcl-xL-Sensitive Intrinsic Apoptosis Pathway

“…Members of the Bcl-2 protein family include the proapoptosis protein Bax-␣ and the antiapotosis proteins Bcl-2 and Bcl-xL (reviewed in references 41 and 6, respectively). Induction of Bax-␣ expression in neurons is associated with apoptosis (4,9,12,31,32,40,63,67). In contrast, induction of Bcl-2 or Bcl-xL expression may prevent neuronal apoptosis induced by various mitochondrial-mediated insults, including NMDA receptor overactivation (2,7,60,68).…”