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N-(1-pyrenyl) maleimide induces bak oligomerization and mitochondrial dysfunction in Jurkat Cells.

Huang PR, Hung SC, Pao CC, Wang TC - Biomed Res Int (2015)

Bottom Line: Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis.Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak.Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan.

ABSTRACT
N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

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NPM induces oligomerization of Bak and Bax. (a) Expression of Bax and Bak in Jurkat and HL60 cells. The cell lysates of Jurkat and HL60 cells were analyzed for the expression of Bax and Bak by Western blot. The effect of NPM on the oligomerization of Bak and Bax in Jurkat (b) and HL60 (c) cells was analyzed by treating the cells with NPM at 0.5–1 μM or with staurosporine (STS) at 500 nM for 3 h. The treated cells were assayed for the presence of Bax and Bak oligomers as described in Section 2. Data shown were from a representative of two independent experiments that gave similar results. Actin served as a loading control. BMH, bismaleimidohexane.
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fig3: NPM induces oligomerization of Bak and Bax. (a) Expression of Bax and Bak in Jurkat and HL60 cells. The cell lysates of Jurkat and HL60 cells were analyzed for the expression of Bax and Bak by Western blot. The effect of NPM on the oligomerization of Bak and Bax in Jurkat (b) and HL60 (c) cells was analyzed by treating the cells with NPM at 0.5–1 μM or with staurosporine (STS) at 500 nM for 3 h. The treated cells were assayed for the presence of Bax and Bak oligomers as described in Section 2. Data shown were from a representative of two independent experiments that gave similar results. Actin served as a loading control. BMH, bismaleimidohexane.

Mentions: Bax and Bak play a critical role in triggering mitochondria-mediated apoptosis [16, 17]. Since the Jurkat (clone E6.1) cells used in this study do not express Bax ([14] and Figure 3(a)) due to frameshift mutation resulting in premature termination of translation [18], we examined whether or not oligomerization of Bak is involved in the NPM-induced apoptosis. To address this issue, Jurkat cells were exposed to varying concentrations of NPM or 500 nM of staurosporine (as a positive control for inducing Bak oligomerization) for 3 h and then treated with membrane-permeable protein cross-linker BMH. As shown in Figure 3(b), NPM induced the formation of Bak homodimer (52 kDa), but not higher order Bak oligomers, in a concentration-dependent manner in Jurkat cells. To address if the lack of higher Bak oligomerization was unique to the Jurkat cells used in this study, we examined the induction of Bax and Bak oligomerization in HL60 cells. As shown in Figure 3(c), NPM and staurosporine induced the formation of higher oligomers of Bak and Bax in HL60 cells.


N-(1-pyrenyl) maleimide induces bak oligomerization and mitochondrial dysfunction in Jurkat Cells.

Huang PR, Hung SC, Pao CC, Wang TC - Biomed Res Int (2015)

NPM induces oligomerization of Bak and Bax. (a) Expression of Bax and Bak in Jurkat and HL60 cells. The cell lysates of Jurkat and HL60 cells were analyzed for the expression of Bax and Bak by Western blot. The effect of NPM on the oligomerization of Bak and Bax in Jurkat (b) and HL60 (c) cells was analyzed by treating the cells with NPM at 0.5–1 μM or with staurosporine (STS) at 500 nM for 3 h. The treated cells were assayed for the presence of Bax and Bak oligomers as described in Section 2. Data shown were from a representative of two independent experiments that gave similar results. Actin served as a loading control. BMH, bismaleimidohexane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4302375&req=5

fig3: NPM induces oligomerization of Bak and Bax. (a) Expression of Bax and Bak in Jurkat and HL60 cells. The cell lysates of Jurkat and HL60 cells were analyzed for the expression of Bax and Bak by Western blot. The effect of NPM on the oligomerization of Bak and Bax in Jurkat (b) and HL60 (c) cells was analyzed by treating the cells with NPM at 0.5–1 μM or with staurosporine (STS) at 500 nM for 3 h. The treated cells were assayed for the presence of Bax and Bak oligomers as described in Section 2. Data shown were from a representative of two independent experiments that gave similar results. Actin served as a loading control. BMH, bismaleimidohexane.
Mentions: Bax and Bak play a critical role in triggering mitochondria-mediated apoptosis [16, 17]. Since the Jurkat (clone E6.1) cells used in this study do not express Bax ([14] and Figure 3(a)) due to frameshift mutation resulting in premature termination of translation [18], we examined whether or not oligomerization of Bak is involved in the NPM-induced apoptosis. To address this issue, Jurkat cells were exposed to varying concentrations of NPM or 500 nM of staurosporine (as a positive control for inducing Bak oligomerization) for 3 h and then treated with membrane-permeable protein cross-linker BMH. As shown in Figure 3(b), NPM induced the formation of Bak homodimer (52 kDa), but not higher order Bak oligomers, in a concentration-dependent manner in Jurkat cells. To address if the lack of higher Bak oligomerization was unique to the Jurkat cells used in this study, we examined the induction of Bax and Bak oligomerization in HL60 cells. As shown in Figure 3(c), NPM and staurosporine induced the formation of higher oligomers of Bak and Bax in HL60 cells.

Bottom Line: Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis.Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak.Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan.

ABSTRACT
N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

Show MeSH
Related in: MedlinePlus