Skip Navigation


Carcinogenesis Advance Access originally published online on February 6, 2008
Carcinogenesis 2008 29(4):729-737; doi:10.1093/carcin/bgn036
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/4/729    most recent
bgn036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Park, S. E.
Right arrow Articles by Yoo, Y. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, S. E.
Right arrow Articles by Yoo, Y. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Interactions of acetylcholinesterase with caveolin-1 and subsequently with cytochrome c are required for apoptosome formation

Sang Eun Park1,3, Seung Hun Jeong1, Soo-Bog Yee1, Tae Hyun Kim1, Young Hwa Soung1, Nam Chul Ha2, Nam Deuk Kim3, Jae-Yong Park4, Hae Rahn Bae5, Bong Soo Park6, Hye Jeong Lee7 and Young Hyun Yoo1,*

1 Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Medical Science Research Center, Busan 602-714, South Korea
2 Department of Manufacturing Pharmacy (BK21 Program)
3 Department of Pharmacy (BK21 Program), The Graduate School, Pusan National University, Busan 609-735, South Korea
4 Department of Physiology, Institute of Health Science and Medical Research Center for Neural Dysfunction, Gyeongsang National University School of Medicine, Jinju 660-751, South Korea
5 Department of Physiology, Dong-A University College of Medicine and Medical Science Research Center, Busan 602-714, South Korea
6 Department of Oral Anatomy and Cell Biology, College of Dentistry, Pusan National University, Busan 602-735, South Korea
7 Department of Pharmacology, Dong-A University College of Medicine and Medical Science Research Center, Busan 602-714, South Korea

* To whom correspondence should be addressed. Tel: +82 51 240 2926; Fax: +82 51 241 3767; Email: yhyoo{at}dau.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Acetylcholinesterase (AChE) is emerging as an important component in leading to apoptosis. Our previous study demonstrated that silencing of the AChE gene blocked the interaction between cytochrome c and apoptotic protease-activating factor-1 (Apaf-1) in etoposide-induced apoptosis of HT-29 cells. We undertook this study to further dissect the molecular role of AChE in apoptosome formation. The present study elicited that small interfering RNA (siRNA) to cytochrome c gene blocked the interaction of AChE with Apaf-1, whereas siRNA to Apaf-1 gene did not block the interaction of AChE with cytochrome c, indicating that the interaction of AChE with cytochrome c is required for the interaction between cytochrome c and protease-activating factor-1. We further observed that AChE is localized to caveolae via interacting with caveolin-1 during apoptosis and that the disruption of caveolae prevented apoptosome formation. These data indicate that the interactions of AChE with caveolin-1 and subsequently with cytochrome c appear to be indispensable for apoptosome formation.

Abbreviations: AChE, acetylcholinesterase; Apaf-1, apoptotic protease-activating factor-1; EGFP, enhanced green fluorescent protein; LMB, leptomycin B; MβCD, methyl-β-cyclodextrin; siRNA, small interfering RNA


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Cholinesterase is a member of the serine hydrolase family using a serine residue at their active site (1). Other than cleaving acetylcholine, it has been revealed that this enzyme may also be active in peptide hydrolysis (2,3), although some reservations regarding this activity have been raised (4). In addition, these enzymes have also been shown to exhibit aryl acylamidase activity (5). There are two major types of cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase. Since butyrylcholinesterase has no known specific natural substrate and its mutation does not result in significant physiological consequences, the function of butyrylcholinesterase remains a puzzle. In contrast, AChE is known to hydrolyze acetylcholine at cholinergic synapses as well as at neuromuscular junctions (6).

Although the key role of AChE is terminating neurotransmission at cholinergic synapses, it is also expressed in tissues devoid of cholinergic responses (1) and several types of hematopoietic cells including erythrocytes and megakaryocytes (7,8). Several studies show abnormality of the AChE gene in several tumors and agricultural use of AChE inhibitors is known to induce several tumors (9). These findings indicated its potential functions beyond neurotransmission.

Recently, the increasing evidence has shown that AChE may be involved in apoptosis. A study demonstrated the presence of AChE activity in various types of apoptotic cells (10). The study also showed that not only pharmacological inhibitors of AChE prevented apoptosis but also blocking the expression of AChE with antisense inhibited apoptosis. Another recent study documented that etoposide or excisanin A-induced expression of AChE messenger RNA and protein in SW620 colon cancer cell line was blocked by a specific inhibitor of stress-activated protein kinase/c-jun N-terminal kinase and small interfering RNA (siRNA) directed against c-jun N-terminal kinase 1/2. The study also showed that transfection with adenovirus-mediated dominant-negative c-jun blocked the upregulation of AChE expression. These results suggest that AChE expression may be mediated by the activation of c-jun N-terminal kinase pathway during apoptosis through a c-Jun-dependent mechanism (11).

We reported previously that silencing of the AChE gene prevented etoposide-induced apoptosis of HT-29 human colon adenocarcinoma cells by blocking the interaction between apoptotic protease-activating factor-1 (Apaf-1) and cytochrome c, which suggested the pivotal role of AChE in apoptosome formation (12). However, detailed molecular mechanism explaining the function of AChE in apoptosome formation is remained largely unknown.

We undertook this study to further dissect the molecular role of AChE in apoptosome formation. The present study demonstrates that AChE interacts with caveolin-1 and subsequently cytochrome c in cells undergoing apoptosis, which interactions appear to be indispensable for apoptosome formation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Apoptosis systems
Most experiments to dissect the molecular role of AChE in apoptosome formation were mainly conducted by employing 40 µg/ml etoposide-induced apoptosis in HT-29 cells. In addition, several other apoptosis systems were also used. Apoptosis types tested are as follows: HT-29 cells treated with 1 mM sulindac for 48 h, with 2.5 µM thapsigargin for 24 h or with 100 µM LY294002; a Phosphoinositide-3-kinase (PI3K) inhibitor for 48 h; Raw264.7 cells treated with a nitric oxide donor SIN-1 at 1 mM for 24 h; SK-MEL-5 cells treated with 10 µg/ml etoposide for 48 h; Malme-3M cells treated with 50 µg/ml etoposide for 24 h; TE671 cells treated with 50 µg/ml etoposide for 24 h; U373MG human brain tumor cells treated with 40 µg/ml etoposide for 24 h; interleukin-3 deprived (murine) bone marrow-derived mast cells and primary cultured rat articular chondrocytes treated with 1.5 mM sodium nitroprusside, a nitric oxide donor, or infected with 100 multiplicity of infection adenoviral TRAIL for 72 h.

siRNA transfection
Twenty-one nucleotide RNA with 3'-dTdT overhangs was synthesized by Dharmacon Research (Lafayette, CO) in the ‘ready-to-use’ option. Sense sequences for AA-N19 messenger RNA targets are as follows: AChE sequence 1, 5'-GCTACGAGATCGAGTTCAT-3'; AChE sequence 2, 5'-CCAATATGTGGACACCCTA-3'; AChE sense sequence 3, 5'-AATATGTGGACACCCTATA-3'; Apaf-1 sense sequence 1, 5'-CATGATTAGTGATGGATTT-3'; Apaf-1 sense sequence 2, 5'-CAGAAGCTCTCCAAATTGA-3'; Apaf-1 sense sequence 3, 5'-ATTGAAAGGTGAACCAGGA-3'; cytochrome c sense sequence 1, 5'-GAGAAAGGCAAGAAGATTT-3'; cytochrome c sense sequence 2, 5'-CACTGATGGAGTATTTGGA-3' and cytochrome c sense sequence 3, 5'-TGGGGAGAGGATACACTGA-3'. As a negative control, the same nucleotides were scrambled to form a non-genomic combination (controlled by basic local alignment search tool). Transfection of siRNA was performed by using siPORT Amine (Ambion, Austin, TX) and Opti-MEM media according to the manufacturer's recommendations. Cells grown to a confluency of 40–50% in six-well plates were transfected with 1 nM final siRNA concentration per well. Twenty-four hours after transfection, apoptotic stimuli were applicated. Among three sense sequences of each target, every sequence 1 showed highly efficient silencing effect. Thus, this single sequence was used for following assays.

Plasmid construction, primers and visualization
Human full-length AChE complementary DNA (accession number: NM_000665 [GenBank] ) was purchased from 21C Frontier Human Gene Bank (21C Frontier Human Gene Bank, Taejeon, Korea) and cloned into pDS_XB-EGFP destination vectors (a gift from Dr Tobias Meyer, Stanford University) by Gateway Cloning System (Invitrogen, Carlsbad, CA). The N-terminal primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCATGAGGCCCCCGCAGTGTCTG-3' was used to introduce an attB1 sequence (underlined) followed by a Kozak sequence (bold) upstream of the coding sequences of AChE. Similarly, the C-terminal primer 5'-GGGG ACCACTTTGTACAAGAAAGCTGGGTTCAGGTCTGAGCAGCGATCCTG-3' was used to introduce attB2 sequences immediately after the last amino acid of these two genes. The full-length AChE gene was amplified on a thermal cycler using DNA polymerase, HiPi Plus PCR premix (Elpis-Biotech, Taejeon, Korea). Polymerase chain reaction products were cloned into pDONR207 vectors using the Gateway Cloning System (Invitrogen). Sequence identity was confirmed by dideoxy sequencing analysis (Macrogen, Seoul, Korea). These entry clones were then added to pDS_XB-EGFP destination vectors in the presence of LR clonase (Invitrogen), and the resulting C-terminal enhanced green fluorescent protein (EGFP)-tagged expression vector (AChE–EGFP) was selected.

To visualize the localization of green fluorescence protein (GFP) fusion AChE protein, HT-29 cells were plated on poly-L-lysine-coated coverslips at a density of 5 x 103 per well (which were cultured in six-well plates) and tranfected by Lipofectamine 2000 reagent (Invitrogen) with expression vectors, AChE–EGFP. Twenty-four hours after transfection, cells were treated with 40 µg/ml etoposide for 0–72 h. Intracellular localization of AChE was analyzed according to the EGFP fluorescent detection method under Zeiss LMS 510 laser scanning confocal microscope (Göettingen, Germany).

Cell viability assay
Cell viability was determined by the Vi-Cell (Beckman Coulter, Fullerton, CA) cell counter that performs an automated trypan blue exclusion assay.

Confocal immunofluorescence microscopic analysis
Cells were seeded on coverslips at a density of 5 x 103 per well, fixed in 3% paraformaldehyde, incubated in primary antibody and fluorescent-tagged secondary antibody. In addition, the nuclei were visualized by Hoechst 33342. Fluorescent images were also observed and analyzed under Zeiss LSM 510 laser scanning confocal microscope.

Quantification of colocalization
Quantification of colocalization was performed using LSM 510 colocalization software program. Briefly, background was subtracted from the images, and the percentage of overlapping pixels between AChE and caveolin-1 or other marker pixels was determined. Eight cells from three experiments were analyzed for n = 24. All data were analyzed by t-test.

Flow cytometric analysis
Cells were trypsinized, washed with phosphate-buffered saline and fixed in 75% ethanol at 4°C for 30 min. Prior to analyses, cells were again washed with phosphate-buffered saline, suspended in cold propidium iodide (Sigma, St Louis, MO) solution and incubated at room temperature in the dark for 30 min. Flow cytometry analyses were performed on a flow cytometry system (Becton Dickinson, San Jose, CA).

Antibodies and antibody array screening
Antibodies against apoptosome molecules used for making the antibody arrays and for the immunoprecipitations were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies (0.5 mg each) were immobilized on polyvinylidene difluoride membranes (5 x 2 cm) at predetermined positions. The antibody array membranes were then incubated with whole-cell lysates from HT-29 cells treated or untreated with 40 µg/ml etoposide for 40 h. After incubation for 2 h, the membranes were blotted with horseradish peroxidase-conjugated anti-AChE polyclonal antibody (against AChE C-terminal; Santa Cruz Biotechnology) for an additional 2 h and followed by enhanced chemiluminescence detection.

Western blot analysis and coimmunoprecipitation
These procedures were performed as described by Park et al. (12). Coimmunoprecipitation assay was reciprocally conducted.

Molecular weight cutoff study
Cell lysate was loaded on a Vivaspin 300 000 MWCO column (Vivascience, Hannover, Germany) and centrifuged at 20 000g. Lower fraction containing monomeric-free Apaf-1 (~130 kDa) and upper fraction containing oligomerized Apaf-1 (300–400 kDa) were collected and aliquots of 50 µl from each fraction were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by western blotting with Apaf-1 antibody.

Statistical analysis
Data are expressed as means ± SDs. Statistical significance of differences was determined by the paired Kruskal–Wallis non-parametric test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
AChE plays a critical role in apoptosome formation
To generalize the involvement of AChE in apoptosome formation, we here first examined whether upregulation of AChE protein is observed in a variety of apoptosis system. Our western blot analysis demonstrated the upregulation of AChE in all types of apoptotic cells tested (supplementary Figure S1A is available at Carcinogenesis Online). We next examined whether the prevention of the interaction between Apaf-1 and cytochrome c by siRNA directed against AChE is a commonly occurring event in apoptosis. Our data show that siRNA directed against AChE prevented apoptosome formation in all types of apoptosis models we tested (Figure 1A).


Figure 1
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. AChE plays a critical role in apoptosome formation. (A) siRNA to AChE gene (si AChE) prevents the interaction between Apaf-1 and cytochrome c, e.g. apoptosome formation, in various types of apoptosis systems. Apoptosis types tested include HT-29 cells treated with 1 mM sulindac for 48 h, with 2.5 µM thapsigargin for 24 h or with 100 µM LY294002, a PI3K inhibitor for 48 h; Raw264.7 cells treated with a nitric oxide donor SIN-1 at 1 mM for 24 h; Malme-3M cells treated with 50 µg/ml etoposide for 24 h and TE671 cells treated with 50 µg/ml etoposide for 24 h. (B) An antibody array indicates that AChE interacts with Apaf-1, caspase-3, caspase-9, cIAP-1, cIAP-2 and cytochrome c. Antibodies immobilized on each division of polyvinylidene difluoride membranes are as follows: (1) Apaf-1; (2) c-IAP-1; (3) c-IAP-2; (4) cytochrome c; (5) p53; (6) Hsp 70; (7) caspase-3 and (8) caspase-9. (C) Coimmunoprecipitation also shows the interaction between AChE with these apoptosome constituents in HT-29 cells treated with 40 µg/ml etoposide for 40 h. (D) Western blotting of upper (U) and lower (L) fractions collected with a molecular weight cutoff method shows that silencing of AChE gene followed by 40 µg/ml etoposide treatment for 40 h prevented the oligomerization of Apaf-1. SCR, scrambled control siRNA.

 
We next tried to reveal the interaction of AChE with various apoptosome constituents by antibody array and coimmunoprecipitation assays. Our data show that AChE interacts with apoptosome constituents, such as Apaf-1, cytochrome c, c-IAP-1, c-IAP-2, caspase-3 and caspase-9 in etoposide-induced apoptosis of HT-29 cells (Figure 1B, supplementary Figure S1B is available at carcinogenesis Online and Figure 1C). Similar results were observed in other several types of apoptosis (supplementary Figure S1C is available at Carcinogenesis Online).

We next examined whether AChE plays a role in the oligomerization of Apaf-1. We undertook molecular weight cutoff study. Lower and upper fractions on 300 000 MWCO column should contain monomeric free Apaf-1 (~130 kDa) and oligomerized Apaf-1, respectively. Our molecular weight cutoff study shows that siRNA to AChE gene prevented etoposide-induced oligomerization of Apaf-1 (Figure 1D).

Interaction between AChE and cytochrome c is required for the assembly of AChE/cytochrome c/Apaf-1 three-component complex in etoposide-treated HT-29 cells
We demonstrated before that the formation of the AChE/cytochrome c/Apaf-1 complex is required for apoptosome formation. We in the present study tried to unveil which interaction among these three components is critical for apoptosome formation. Our data indicate that the silencing of Apaf-1 gene efficiently downregulated Apaf-1, resulting in the prevention of apoptosis (Figure 2A). Noticeably, the interaction between AChE and cytochrome c was maintained throughout the tested period. The presence of the interaction of AChE with cytochrome c, under this condition, was also observed not only in Apaf-1-negative SK-MEL-5 cells undergoing apoptosis after etoposide treatment but also in Apaf-1–/– knockout cells (kindly provided by Dr David Vaux, The Walter and Eliza Hall Institute, Parkville, Victoria, Australia) undergoing apoptosis after interleukin-3 deprivation (13, Figure 2B).


Figure 2
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Interaction between AChE and cytochrome c is required for the interaction between cytochrome c and Apaf-1 in etoposide-treated HT-29 cells. (A) Silencing of the Apaf-1 gene efficiently downregulated the expression of Apaf-1 and significantly prevented the reduction of cell viability induced by 40 µg/ml etoposide. Viability was determined by a cell counter performing an automated trypan blue exclusion assay. Statistically significance: **P < 0.01. (B) AChE maintains the interaction with cytochrome c irrespective of the silencing of Apaf-1 gene by si Apaf-1. Although si Apaf-1 blocks the interaction between AChE and Apaf-1, it does not prevent the interaction of AChE with cytochrome c. The presence of the interaction of AChE with cytochrome c is also observed not only in Apaf-1-negative SK-MEL-5 cells treated with 10 µg/ml etoposide but also in interleukin-3-deprived Apaf-1–/– knockout cells. Western blot of immunoprecipitated AChE from the cell lysates obtained 24 h after application of apoptotic stimuli is presented as a positive control. (C) Silencing of cytochrome c gene efficiently downregulated the expression of cytochrome c and significantly prevented the reduction of cell viability induced by 40 µg/ml etoposide. Viability was determined by a cell counter performing an automated trypan blue exclusion assay. Statistically significance: **P < 0.01. Data are presented as mean ± SD (n = 4). (D) siRNA to cytochrome c gene (si Cyt c) prevents the interactions of AChE not only with cytochrome c but aslo with Apaf-1. IgG is shown as a loading control. Western blot of immunoprecipitated Apaf-1 or AChE from the cell lysates obtained 24 h after etoposide treatment is presented as positive controls. (E) Western blotting of upper (U) and lower (L) fractions collected with a molecular weight cutoff method shows that silencing cytochrome c gene by si Cyt c followed by 40 µg/ml etoposide treatment for 40 h prevented the oligomerization of Apaf-1. PC, positive control.

 
On the other hand, the silencing of cytochrome c gene efficiently downregulated cytochrome c, resulting in the prevention of apoptosis (Figure 2C and supplementary Figure S2A is available at carcinogenesis Online). Importantly, the silencing of cytochrome c gene prevented the interactions of AChE not only with cytochrome c but also with Apaf-1 (Figure 2D). Our molecular weight cutoff study also showed that siRNA to cytochrome c gene prevented the oligomerization of Apaf-1 (Figure 2E). These data indicate that the interaction between AChE and cytochrome c is required for the interaction between cytochrome c and Apaf-1. We also observed that the silencing of cytochrome c gene prevented the interaction between AChE and Apaf-1 in several other apoptosis systems (supplementary Figure S2B is available at carcinogenesis Online).

AChE shuttles between the nucleus and the cytoplasm during apoptosis
Laser scanning confocal microscopy at different time points using the fusion protein AChE–GFP in HT-29 cells treated with etoposide showed dynamic subcellular localization of fusion protein AChE–GFP. The fusion protein was observed out of the nucleus in control cells (0 h). However, etoposide treatment induced the shuttling of AChE. During the early phases of apoptosis (1–4 h), AChE was first translocated into nucleus. AChE was then observed out of the nucleus to the cytosol (8–16 h), forming a ring structure around the nucleus. At the later phases, AChE was localized on the periphery of cells (24–40 h). At the end time point of apoptosis (72 h), in which most cells showed the phenotype of apoptotic body with disrupted nucleus, AChE appeared to be present on the nucleus as well as in the cytosol (Figure 3A).


Figure 3
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. AChE shuttles between the nucleus and the cytoplasm during apoptosis. (A) Confocal microscopy shows a time-sequenced shuttling of GFP–AChE between the nucleus and the cytoplasm after treatment with 40 µg/ml etoposide. AChE, out of the nucleus in control cells (0 h), was first translocated into nucleus (1–4 h) and then out of the nucleus to the cytosol (8–16 h). At the later phases, AChE was localized on the periphery of cells (24–40 h). At the end time point of apoptosis (72 h), AChE appeared to be present on the nucleus as well as in the cytosol. (B) Pretreatment of LMB induced accumulation of AChE on the nucleus and prevented the induction of apoptosis. Control (a), Whereas 40 µg/ml etoposide treatment for 40 h induced the localization of AChE to the periphery of cells (b), pretreatment of 5 nM LMB for 3 h followed by 40 µg/ml etoposide treatment for 40 h induced accumulation of AChE in the nucleus (c). Flow cytometry shows that pretreatment of 5 LMB prevented the induction of DNA hypoploidy. The arrow indicates the sub-G1 apoptotic population. Scale bar, 10 µm.

 
We then examined whether blocking nuclear export of AChE prevented apoptosis or not. AChE was observed on the periphery of cells treated with etoposide for 40 h. However, in cells pretreated with leptomycin B (LMB), an inhibitor of nuclear export receptor chromosome region maintenance 1, AChE was observed on the nucleus, indicating that LMB efficiently induced accumulation of AChE on the nucleus. Noticeably, DNA hypoploidy determination by flow cytometry elucidated that the induction of apoptosis was prevented by LMB pretreatment (Figure 3B). These data suggest that blocking nuclear export of AChE prevents apoptosis.

AChE colocalizes with caveolin-1 during apoptosis
We further determined subcellular localization of AChE by double immunofluorescence utilizing several cell organelle markers. Our data obtained by employing an antibody directed against the endoplasmic reticulum-associated protein disulfide isomerase and a lysosomal fluorescent probe LysoTracker DND-99 showed that AChE was neither localized on endoplasmic reticulum nor on lysosome. Rather, immunofluorescent staining to AChE appeared uneven and punctuate over the plasma membrane (Figure 4A).


Figure 4
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. AChE colocalizes with caveolin-1. (A) Confocal microscopy using cell organelle markers shows the subcellular location of AChE in HT-29 cells treated with 40 µg/ml etoposide for 40 h. Confocal microscopy using antiprotein endoplasmic reticulum (ER)-associated protein disulfide isomerase antibody to detect endoplasmic reticulum or fluorescent probes LysoTracker DND-99 to detect lysosome demonstrates that AChE is neither localized on endoplasmic reticulum nor on lysosome. The lowest confocal microscopy using FM 1-43FX to detect plasma membrane shows that AChE appeared uneven and punctuate over the plasma membrane. Hoechst 33342 to detect the nucleus. LT, LysoTracker; PM, plasma membrane. Quantification of confocal images demonstrates that colocalization of AChE with plasma membrane is significantly enhanced in treated cells (40 h) compared with the control (0 h). **P < 0.01. (B) Confocal microscopy shows the colocalization of AChE with caveolin-1 in HT-29 cells treated with 40 µg/ml etoposide. Quantification of confocal images demonstrates that colocalization of AChE with caveolin-1 is significantly enhanced in treated cells (40 h) compared with the control (0 h). **P < 0.01. Coimmunoprecipitation shows a time-sequenced interaction of AChE with caveolin-1 in HT-29 cells treated with 40 µg/ml etoposide. IgG is shown as a loading control. Hoechst 33342 to detect the nucleus. (C) Confocal microscopy also shows the colocalization of AChE with caveolin-1 in HT-29 cells treated with 100 µM LY294002 for 24 h, with 1 mM sulindac for 48 h or with 2.5 µM thapsigargin for 24 h. Scale bar, 10 µm. Quantification of confocal images demonstrates that colocalization of AChE with caveolin-1 is significantly enhanced in treated cells (40 h) compared with the control (0 h) of each treatment. **P < 0.01.

 
Thus, we examined whether AChE was localized to a specific plasma membrane compartment, caveolae, via its interaction with caveolin-1. As shown in the upper panel of Figure 4B, confocal microscopic study demonstrated the colocalization of AChE with caveolin-1. The merged image obtained by anti-AChE antibody together with an anti-caveolin-1 antibody clearly revealed yellow-orange areas, resulting from the overlap of green and red fluorescences, which corresponded to nearly complete colocalization, suggesting their interaction. The validity of this interaction was confirmed by coimmunoprecipitation assay (lower panel of Figure 4B and supplementary Figure S3A is available at carcinogenesis Online). This colocalization was also evidently observed in other types of apoptosis (Figure 4C).

Interaction of AChE with caveolin-1 is indispensable for apoptosome formation
We next asked whether the interaction of AChE with caveolin-1 is indispensable for apoptosome formation. Thus, we used methyl-β-cyclodextrin (MβCD) which not only disrupts caveolae structure but also reduces caveolin-1 expression. We observed that MβCD pretreatment prevented the reduction of viability of cells (Figure 5A). Confocal microscopy showed that AChE was accumulated not on the periphery but in the cytosol of cells pretreated with MβCD in which caveolin-1 was hardly detected (Figure 5B). Moreover, MβCD pretreatment prevented not only the interaction between AChE and caveolin-1 but also the interaction of cytochrome c with AChE or with Apaf-1 (Figure 5C and supplementary Figure S3B is available at carcinogenesis Online). Noticeably, our molecular weight cutoff study revealed that MβCD pretreatment prevented the oligomerization of Apaf-1 (Figure 5D). In addition, we observed that silencing of cytochrome c gene did not prevent the interaction between AChE and caveolin-1 (Figure 5E and supplementary Figure S3C is available at carcinogenesis Online). To this end, we examined the relevance of apoptosome localization at the caveolae. Our coimmunoprecipitation data show that neither cytochrome c nor Apaf-1 interacts with caveolin-1 in etoposide-treated HT-29 cells, which indicates that apoptosome does not localize on the caveolae (supplementary Figure S3D is available at carcinogenesis Online). Taken together, our data suggest that the interaction between AChE and caveolin-1 precedes apoptosome formation, indicating that the interaction of AChE with caveolin-1 is indispensable for obtaining its capability pertaining to apoptosome formation.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Interaction of AChE with caveolin-1 is indispensable for apoptosome formation. (A) MβCD (100 mg) was dissolved in 2 ml of distilled water prior to the experiments. Pretreatment of HT-29 cells with 0.5 mg/ml MβCD for 1/12, 1, 2 or 24 h followed by 40 µg/ml etoposide treatment for 40 h prevents cell death. Viability was determined by a cell counter performing an automated trypan blue exclusion assay. Statistically significance: *P < 0.05; **P < 0.01. Data are presented as mean ± SD (n = 4). (B) Confocal microscopy shows that pretreatment of HT-29 cells with 0.5 mg/ml MβCD for 24 h followed by 40 µg/ml etoposide treatment for 40 h induces accumulation of AChE in the cytosol. Hoechst 33342 to detect the nucleus. Scale bar, 10 µm. Quantification of confocal images demonstrates that MβCD significantly reduced colocalization of AChE with caveolin-1 in treated cells compared with the experimental control. **P < 0.01. (C) Coimmunoprecipitation shows that pretreatment of HT-29 cells with 0.5 mg/ml MβCD for 1/12, 1, 2 or 24 h followed by 40 µg/ml etoposide treatment for 40 h prevents the interactions of AChE with caveolin-1 or cytochrome c and of cytochrome c with Apaf-1. IgG is shown as a loading control. (D) Western blotting of upper (U) and lower (L) fractions collected with a molecular weight cutoff method shows that MβCD pretreatment for 24 h followed by etoposide treatment for 40 h prevents the oligomerization of Apaf-1. (E) Coimmunoprecipitation shows that silencing of cytochrome c gene does not prevent the interaction between AChE and caveolin-1. IgG is shown as a loading control.

 
We then employed caveolin-negative cell lines, LNCaP and PC-3 cells, to prove the role of the interaction between AChE and caveolin-1 in apoptosome formation. Although AChE was also upregulated in etoposide-treated LNCaP and PC-3 cells (Figure 6A), no interaction of AChE and cytochrome c, AChE and Apaf-1 or Apaf-1 and cytochrome c is observed in these cells (Figure 6B and supplementary Figure S3E is available at carcinogenesis Online). Accordingly, the interaction between AChE and caveolin-1 appears to be indispensable for apoptosome formation.


Figure 6
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Apoptosome is not formed in caveolin-1-negative cell lines, LNCaP and PC3 cells, treated with etoposide. (A) Confocal microscopy and western blotting show that the expression level of AChE was upregulated. Scale bar, 10 µm. (B) Coimmunoprecipitation assay shows absence of the interactions of AChE with Apaf-1, caveolin-1 and cytochrome c and of Apaf-1 with cytochrome c. IgG is shown as a loading control. Western blot of immunoprecipitated Apaf-1, AChE or cytochrome c from the cell lysates obtained 24 h after etoposide treatment is presented as a positive control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
In our previous study, we reported that AChE exerted its pivotal role in apoptosis by participating in the formation of apoptosome (12). To dissect further the molecular role of AChE in apoptosome formation, we first tried to corroborate that the upregulation of AChE is commonly observable in apoptosis. Our western blotting data, in addition to reverse transcription–polymerase chain reaction data from a previous study which demonstrated the expression of AChE messenger RNA in various types of apoptosis (10), suggest that the upregulation of AChE is commonly observable in apoptosis. Furthermore, we demonstrated that siRNA directed against AChE prevented apoptosome formation in all types of apoptosis systems we tested. Therefore, we assume that not only the upregulation of AChE but also the prevention of apoptosome formation by silencing AChE gene appears to be a commonly observable phenomenon in apoptosis.

The Apaf-1 apoptosome is a multisubunit caspase-activating scaffold that is assembled in response to various forms of apoptotic stimuli (14). The release of cytochrome c from the mitochondrial intermembrane space provokes the assembly of the apoptosome, which is both positively and negatively regulated by members of the Bcl-2 family (15). Apaf-1, when bound to cytochrome c, hydrolyzes 2'-deoxyadenosine 5'-triphosphate/adenosine triphosphate and undergoes oligomerization via its CED-4 domains. Simultaneously, the caspase recruitment domain recruits and facilitates the processing of procaspase-9. This complex of cytochrome c, Apaf-1 and caspase-9 is commonly referred to as the apoptosome and this apoptosome formation is a crucial step in death receptor-mediated apoptosis as well as type I apoptosis (16). Several additional proteins in addition to cytochrome c/Apaf-1/caspase-9 are known to be implicated as apoptosome constituents, which include caspase-3, caspase-7, XIAP, Aven, NAC, Bcl-2 and Bcl-XL (17–19). Reconstitution experiments using purified recombinant proteins indicate that the apoptosome is ~1.4 MDa in size, whereas in native cell lysates, Apaf-1 oligomerizes into ~700 kDa complex. However, the specific mechanisms that govern these processes remain unclear. Our data show that AChE not only interacts with various apoptosome constituents but also plays a pivotal role in the oligomerization of Apaf-1. Noticeably, our data indicate that the interaction between AChE and cytochrome c is required for the interaction between cytochrome c and Apaf-1. Thus, we assume that the interaction between AChE and cytochrome c is indispensable for the interaction between cytochrome c and Apaf-1.

Nucleocytoplasmic transport of signal transducers and execution factors may be a crucial and even essential aspect of apoptosis. Several proteins implicated in apoptotic cell death have been shown to migrate in and out of the nucleus following apoptosis induction. Active transport signals have been identified in an increasing number of cellular proteins executing apoptosis and most of proapoptotic nuclear factors are known to be activated in the cytoplasm and to gain access to the nucleoplasm during apoptotic process (20). Accordingly, the nuclear uptake and release of apoptotic factors appear to play important roles in the execution as well as in the initiation of the apoptotic program. The nucleus is also the main target for genotoxic insult. Signals generated in the nucleus by DNA damage have to propagate to all cellular compartments, ensuring the coordinated execution of apoptosis. Thus, the nucleocytoplasmic shuttling of signaling and execution factors is thus an integral part of the apoptotic program.

Previous studies demonstrated that AChE was found in the cytoplasm at the initiation of apoptosis and then in the nucleus or apoptotic bodies upon commitment to cell death (10,21). We here demonstrated the localization of AChE out of the nucleus in healthy cells, which suggests that the nuclear export of AChE predominates over the import process. Etoposide treatment to HT-29 cells is probably to alter this equilibrium. After etoposide treatment, AChE shuttled between the nucleus and the cytoplasm during apoptosis. Noticeably, LMB induced accumulation of AChE on the nucleus, which results in the prevention of apoptosis. These findings indicate that nuclear export of AChE via exportin-1 is an indispensable step in apoptosis. AChE is known to have a nucleus localization signal (22). Thus, it could translocate onto nucleus on which it seems to play a certain role contributing to the apoptotic process. Although a previous study suggested that AChE on the nucleus may participate in the modulation of nuclear components leading to chromatin condensation and fragmentation (10), it remains an open question why AChE first enter to nucleus and then export into cytoplasm during apoptosis induction.

Caveolae are specialized invaginated microdomains of the plasma membrane (23), which are composed mainly of cholesterol and sphingolipids. Their associated proteins, the caveolins, are a family of 21–25 kDa integral membrane proteins and the primary structural constituent of caveolae. Currently, the functional roles attributed to caveolae and caveolin-1 are quite diverse, ranging from vesicular transport (transcytosis, endocytosis and potocytosis) and cholesterol homeostasis to the suppression of cell transformation and the regulation of signal transduction (24,25). It has been shown that many signaling molecules directly interact with caveolin-1 through a defined modular protein domain, known as the caveolin-scaffolding domain (residues 82–101) (26). Thus, the ability of caveolin-1 and caveolae to modulate signaling has important implications for the process of cell transformation and tumor formation.

To date, the role of caveolin-1 in apoptosis remains enigmatic. The role of caveolin-1 in apoptosis appears to be antithetic in different systems. A study showed that caveolin-1 overexpression sensitizes fibroblasts to ceramide-induced death through a PI3K-dependent mechanism (27). Another study elicited that caveolin-1 expression sensitizes both NIH-3T3 fibroblasts and T24 bladder carcinoma cells to apoptosis initiated by staurosporine (28). In contrast, disruption of caveolae showed to block interleukin-6- and insulin-like growth factor-1-induced activation of the PI3K/Akt survival signaling pathway (29). It was also depicted that caveolin-1 mediates cell survival by sustaining Akt activation through the binding and inhibition of the serine/threonine protein phosphatases, namely PP1 and PP2A (30). This incongruity of the proapoptotic or antiapoptotic functions of caveolin-1 may be explained by cell-type-specific effects. Alternatively, the disparate effects of caveolin-1 may be due to the use of different apoptotic inducers. Further future studies will be necessary to distinguish between these two possibilities.

Our data indicate that AChE interacts with caveolin-1 in cells undergoing apoptosis. We observed not only disruption of caveolae and downregulation of caveolin-1 prevented apoptosome formation but also apoptosome was not formed in caveolin-negative cells. In addition, we also demonstrated that the interaction between AChE and caveolin-1 is required for the interaction between AChE and cytochrome c. These data indicate that the interaction between AChE and caveolin-1 appears to be indispensable for apoptosome formation. However, we observed that neither cytochrome c nor Apaf-1 interacts with caveolin-1. These data indicate that the interaction between AChE and caveolin-1 precedes apoptosome formation and that AChE obtains its capability pertaining to apoptosome formation via the interaction with caveolin-1. Thus, we assume that the interactions of AChE with caveolin-1 and subsequently cytochrome c in cells undergoing apoptosis appear to precede apoptosome formation.

We propose that regulated subcellular localization of AChE and its interaction with other apoptosis constituents are a crucial and even essential aspect of apoptosis, mainly related with apoptosome formation. However, the relationships between AChE protein and classical apoptotic molecules during apoptosis remain to be elucidated. Improved understanding of this unresolved regulation will yield new insights of general interest in apoptosis biology and identify pathways that might be targeted for the design of anticancer therapeutics.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figures S1–3 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Korea Research Foundation (KRF-2006-J03501).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Small DH, et al. Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease. Neurochem. Int. (1996) 28:453–483.[CrossRef][Web of Science][Medline]
  2. Wright CI, et al. Protease inhibitors and indolamines selectively inhibit cholinesterases in the histopathologic structures of Alzheimer's disease. Ann. N. Y. Acad. Sci. (1993) 695:65–68.[CrossRef][Web of Science][Medline]
  3. Salmon AY, et al. Human erythrocyte but not brain acetylcholinesterase hydrolyses heroin to morphine. Clin. Exp. Pharmacol. Physiol. (1999) 26:596–600.[CrossRef][Web of Science][Medline]
  4. Checler F. Non-cholinergic actions of acetylcholinesterases: a genuine peptidase function or contaminating proteases? Trends Biochem. Sci. (1990) 15:337–338.[Web of Science][Medline]
  5. Jayanthi LD, et al. Cholinesterases exhibiting aryl acylamidase activity in human amniotic fluid. Clin. Chim. Acta (1992) 205:157–166.[CrossRef][Web of Science][Medline]
  6. Taylor P, et al. The cholinesterases: from genes to proteins. Annu. Rev. Pharmacol. Toxicol. (1994) 34:281–320.[CrossRef][Web of Science][Medline]
  7. Zeng WR, et al. Refined mapping of the region of loss of heterozygosity on the long arm of chromosome 7 in human breast cancer defines the location of a second tumor suppressor gene at 7q22 in the region of the CUTL1 gene. Oncogene (1999) 18:2015–2021.[CrossRef][Web of Science][Medline]
  8. Takahashi S, et al. Frequent loss of heterozygosity at 7q31.1 in primary prostate cancer is associated with tumor aggressiveness and progression. Cancer Res. (1995) 55:4114–4119.[Abstract/Free Full Text]
  9. Dich J, et al. Pesticides and cancer. Cancer Causes Control (1997) 8:420–443.[CrossRef][Web of Science][Medline]
  10. Zhang XJ, et al. Induction of acetylcholinesterase expression during apoptosis in various cell types. Cell Death Differ. (2002) 9:790–800.[CrossRef][Web of Science][Medline]
  11. Deng R, et al. Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis. Oncogene (2006) 53:7070–7077.
  12. Park SE, et al. Acetylcholinesterase plays a pivotal role in apoptosome formation. Cancer Res. (2004) 64:2652–2655.[Abstract/Free Full Text]
  13. Ekert PG, et al. Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J. Cell Biol. (2004) 165:835–842.[Abstract/Free Full Text]
  14. Li P, et al. Cytochrome c and dATP-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell (1997) 91:479–489.[CrossRef][Web of Science][Medline]
  15. Kuwana T, et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell (2002) 111:331–342.[CrossRef][Web of Science][Medline]
  16. Rudner J, et al. Type I and type II reactions in TRAIL-induced apoptosis–results from dose-response studies. Oncogene (2005) 24:130–140.[CrossRef][Web of Science][Medline]
  17. Qin H, et al. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature (1999) 399:549–559.[CrossRef][Medline]
  18. Chau BN, et al. Aven, a novel inhibitor of caspase activation, binds Bcl-xL and apaf-1. Mol. Cell (2000) 6:31–40.[CrossRef][Web of Science][Medline]
  19. Bratton SB, et al. Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes. EMBO J. (2001) 20:998–1009.[CrossRef][Web of Science][Medline]
  20. Ferrando-May E. Nucleocytoplasmic transport in apoptosis. Cell Death Differ. (2005) 12:1263–1276.[CrossRef][Web of Science][Medline]
  21. Jin QH, et al. Overexpression of acetylcholinesterase inhibited and promoted apoptosis in NRK cells. Acta Pharmacol. Sin. (2004) 8:1013–1021.
  22. Perry C, et al. Complex regulation of acetylcholinesterase gene expression in human brain tumors. Oncogene (2002) 21:8428–8441.[CrossRef][Web of Science][Medline]
  23. Razani B, et al. Caveolae: from cell biology to animal physiology. Pharmacol. Rev. (2002) 54:431–467.[Abstract/Free Full Text]
  24. Galbiati F, et al. Emerging themes in lipid rafts and caveolae. Cell (2001) 106:403–411.[CrossRef][Web of Science][Medline]
  25. Torres VA, et al. Caveolin-1 controls cell proliferation and cell death by suppressing expression of the inhibitor of apoptosis protein survivin. J. Cell Sci. (2006) 119:1812–1823.[Abstract/Free Full Text]
  26. Couet J, et al. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J. Biol. Chem. (1997) 272:6525–6533.[Abstract/Free Full Text]
  27. Zundel W, et al. Caveolin 1-mediated regulation of receptor tyrosine kinase-associated phosphatidylinositol 3-kinase activity by ceramide. Mol. Cell. Biol. (2000) 20:1507–1514.[Abstract/Free Full Text]
  28. Liu J, et al. Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation. Am. J. Physiol. Cell Physiol. (2001) 280:C823–C835.[Abstract/Free Full Text]
  29. Podar K, et al. Essential role of caveolae in interleukin-6- and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple myeloma cells. J. Biol. Chem. (2003) 278:5794–5801.[Abstract/Free Full Text]
  30. Li L, et al. Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A. Mol. Cell. Biol. (2003) 23:9389–9404.[Abstract/Free Full Text]
Received November 30, 2007; revised January 17, 2008; accepted January 26, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/4/729    most recent
bgn036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Park, S. E.
Right arrow Articles by Yoo, Y. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, S. E.
Right arrow Articles by Yoo, Y. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?