Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis

doi:10.1007/s13238-011-1071-9

Prot Cell

2011, Vol. 2

Issue (7) : 543-553 https://doi.org/10.1007/s13238-011-1071-9 PMID: 21822799

RESEARCH ARTICLE

Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis

Xiao-Xi Guo¹, Hanna Kim², Yang Li¹, Hyungshin Yim², Seung Ki Lee²(

), Ying-Hua Jin¹(

)

1. Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, Jilin University, Changchun 130012, China; 2. Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea

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Abstract

Sequential activation of cyclin-dependent kinases (Cdks) controls mammalian cell cycle. Here we demonstrate that the upregulation of cyclin-dependent kinase 2 (Cdk2) activity coincides with the loss of mitochondrial membrane potential (MMP) in paclitaxel-induced apoptosis. Ectopic expression of the dominant negative Cdk2 (Cdk2-dn) and a specific Cdk2 inhibitor, p21^WAF1/CIP1, effectively suppresses the loss of MMP, the release of cytochrome c, and subsequent activation of caspase-3 in paclitaxel-treated cells. Whereas forced activation of Cdk2 by overexpression of cyclin A dramatically promotes these events. We further show that Cdk2 activation status does not interfere with a procedure that lies downstream of cytochrome c release induced by Bax protein. These findings suggest that Cdk2 kinase can regulate apoptosis at earlier stages than mitochondrial permeability transition and cytochrome c release.

Keywords apoptosis cyclin-dependent kinase 2 cytochrome c release mitochondrial membrane potential paclitaxel

Corresponding Author(s): Lee Seung Ki,Email:sklcrs@plaza.snu.ac.kr (S. K. Lee); Jin Ying-Hua,Email:yhjin@jlu.edu.cn (Y.-H. Jin)

Issue Date: 01 July 2011

Cite this article:

Xiao-Xi Guo,Hanna Kim,Yang Li, et al. Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis[J]. Prot Cell, 2011, 2(7): 543-553.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1071-9
https://academic.hep.com.cn/pac/EN/Y2011/V2/I7/543

Fig.1 Paclitaxel-induced apoptosis in HeLa cells occurs after cell cycle arrest in G/M phase.
(A) HeLa cells were treated with paclitaxel (80 nmol/L) for indicated times and flow cytometric analysis was carried out as described in MATERIALS AND METHODS. (B) The proportion of cell populations in the G/M phase was calculated and is represented by a histogram. (C) The proportion of cell populations in the subG fraction was calculated and is represented by a histogram. (D) Cells stained with Mitocapture were photographed by phase contrast microscopy (×100) (upper panel) or fluorescence microscopy (×100) (lower level). (E) HeLa cells were treated with paclitaxel (80 nmol/L) in the presence or absence of z-VAD-fmk (30 μmol/L) for indicated times. Cell lysates were prepared and the same amounts of proteins of each sample were analyzed for caspase-3 (DEVDase) cleavage activity using cell-free protease assay as described in MATERIALS AND METHODS.

Fig.2 Both cyclin B-Cdc2 and cyclin A-Cdk2 kinase activities are up-regulated during paclitaxel-induced apoptosis.
(A–C) Whole cell extracts were prepared from HeLa cells treated with paclitaxel (80 nmol/L) for indicated times and resolved by SDS-PAGE and analyzed by immunoblotting using specific antibodies against PARP, cyclin A, Cdk2, cyclin B, cyclin E, Cdc2 and β-actin. Cdk2 (B) and Cdc2 (C) immune complex kinase assay was performed using histone H1 as a substrate as described in MATERIALS AND METHODS.

Fig.3 Ectopic expression of Cdk2-dn and p21WAF1/CIP1 suppresses paclitaxel-induced apoptosis, while that of cyclin A accelerates apoptosis.
HeLa cells were cotransfected with 1.5 μg of pCMV, pCMV-cyclin A, pCMV-cdk2-dn, pCMV-p21, or pCMV-Bcl-2 and 0.5 μg of pCMV-GFP. Twenty-four hours after transfection, the cells were induced to undergo apoptosis by incubation with paclitaxel (80 nmol/L) for 10, 14, 18, and 24 h, respectively. (A) Bright-field and GFP fluorescence of the same field (×100) of transfected cells, which were incubated with paclitaxel for 14 h. (B) GFP expressing cells with blebbing or normal morphologies were counted. The average numbers in five different fields from two independent experiments are shown. (C) Caspase-3 activity in transfected cells after treatment with paclitaxel for 14 h was determined using Ac-DEVD-AFC as a substrate. (D) Immunoblotting analysis of the levels of cyclin A, Cdk2-dn, p21, Bcl-2 and β-actin expressed in the transfectants.

Fig.4 Overexpression of Cdk2-dn, or p21 prevents paclitaxel-inducedmitochondrial membrane deporlarization, cytochrome c release, mitochondrial bax upregulation and apoptosis progression.
(A) HeLa cells were cotransfected with 1.5 μg of pCMV, pCMV-cyclin A, pCMV-Cdk2-dn, pCMV-p21 or pCMV-Bcl-2 and 0.5 μg of pCMV-GFP. Twenty-four hours after transfection, the cells were induced to undergo apoptosis by incubation with paclitacel (80 nmol/L). After 14 h of incubation with paclitaxel, the cells were stained with MitoCapture (CalBiochem) and examined by fluorescence microscopy (Olympus). Upper level: red fluorescence indicating mitochondrion staining; middle level: GFP fluorescence, and lower level: bright-field of the same field cells. (B) Transfected GFP expressing cells with normal mitochondria or deporlarized mitochondria (red bar), blebbing or normal morphologies (green bar) were counted. The results show the average numbers in five different fields from two independent experiments. (C) Cytosolic cytochrome c and β-actin were determined by immunoblotting analysis (upper panel), protein levels of cyclin A, Cdk2-dn p21 and Bcl-2 in transfercted cells (lower panel). (D) The protein levels of cytochrome c and β-actin in (C) were densitometically measured and their ratios are presented. (E) Mitochondrial Bax, Bcl-2, and COX Ⅳ were determined by immunoblot analysis.

Fig.5 Overexpression of cyclin A, Cdk2-dn, and p21 does not inhibit Bax-induced PARP cleavage and caspase-3/7 activation.
(A) HeLa cells were cotransfected with 1.5 μg of pCMV, pCMV-Bcl-2, pCMV-cyclin A, pCMV-Cdk2-dn, pCMV-Cdc2-dn, pCMV-p21with or without 0.5 μg of pCMV-Bax. Twenty-four hours after transfection, the cells were harvested and analyzed using specific antibodies against PARP, Bcl-2, cyclin A, Cdk2, Cdc2, p21, Bax, and β-actin. (B) The same amounts of proteins of each sample were analyzed for caspase-3 (DEVDase) cleavage activity using cell-free protease assay as described under MATERIALS AND METHODS.

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