Open Access

The tumor suppressor DAL-1/4.1B and protein methylation cooperate in inducing apoptosis in MCF-7 breast cancer cells

Molecular Cancer20065:4

https://doi.org/10.1186/1476-4598-5-4

Received: 26 August 2005

Accepted: 18 January 2006

Published: 18 January 2006

Abstract

Background

DAL-1 (Differentially Expressed in Adenocarcinoma of the Lung)/4.1B is a member of the protein 4.1 superfamily that has been shown to suppress growth in lung, breast and brain tumor cells. In the case of the caspase-3 deficient MCF-7 breast cancer cells, this growth suppression has been shown to be partially mediated by the induction of apoptosis. However the exact mechanism of action of DAL-1/4.1B is unknown. Recently, protein arginine N-methyltransferase 3 (PRMT3) was identified as a DAL-1/4.1B interacting protein. Protein arginine methyltransferases (PRMTs) posttranslationally methylate the arginine residues of proteins, a modification which has been implicated in the regulation of multiple cellular processes including nuclear-cytoplasmic transport, signal transduction, and transcription.

Results

To investigate the role of protein methylation in cell death induced by DAL-1/4.1B, DAL-1/4.1B-inducible MCF-7 cells were examined for apoptosis and caspase activation in the absence and presence of the protein methylation inhibitor adenosine dialdehyde (AdOX). Flow cytometry analysis revealed that apoptosis was primarily associated with the activation of caspase 8, and inhibition of this activation blocked the ability of DAL-1/4.1B to induce cell death.

Conclusion

These results suggest that protein methylation cooperates with DAL-1/4.1B-associated caspase 8-specific activation to induce apoptosis in breast cancer cells.

Background

Differentially expressed in adenocarcinoma of the lung (DAL-1)/4.1B is a tumor suppressor gene belonging to the Protein 4.1 superfamily [1]. Like other members of this family, DAL-1/4.1B localizes to the cell membrane and contains an N-terminal 4.1/ezrin/radixin/moesin (FERM) domain [2] and spectrin/actin binding sequences. When introduced into DAL-1/4.1B-null lung, breast and meningioma cancer cell lines, this Protein 4.1 family member significantly suppresses growth, in part through the induction of apoptosis [1, 3, 4]. However, the pathways via which DAL-1/4.1B exerts its growth suppressing properties are still poorly understood.

The FERM domain of the founding family member Protein 4.1R has been found to associate with several membrane proteins, including erythrocyte band 3, calmodulin, glycophorin C, p55 and spliceosome-associated pICln [57]. Similarly, merlin/NF2 associates with several transmembrane proteins including CD44 via residues in the N-terminal FERM domain [8, 9]. The interaction of merlin/NF2 with CD44 has been shown to be critical for its growth suppression [8, 10]. Hypothesizing that the unique binding partners for DAL-1/4.1B may help elucidate its mechanism of action as a negative growth regulator, yeast two-hybrid analysis was performed using the 336 residues of DAL-1/4.1B FERM domain and a fetal lung cDNA library. Several strongly associating proteins, including 14-3-3 protein isoforms β, γ and η [11] and protein arginine N-methyltransferase 3 (PRMT3) [12] were identified. PRMT3 and its family members post-translationally form asymmetric ω-NG, NG- (Type I enzymes; PRMT1, 2, 3, 4, and 6) or symmetric w-NG, N'G- (Type II enzymes; PRMT5) dimethylarginine residues on proteins. This protein modification has been shown to regulate transduction of signals to the nucleus, transcription regulation through nuclear receptors, and RNA transport between the nucleus and cytoplasm [1319]

Recently we have reported that DAL-1/4.1B regulates the methylation of substrates by PRMT3 [12] and PRMT5 [20] both in vitro and in cultured cells. Based on these findings, post-translational protein methylation may be one mechanism by which DAL-1/4.1B suppresses growth and induces apoptosis in MCF-7 cells. To address this, DAL-1/4.1B-induced apoptosis and caspase activation were analyzed in both control and hypomethylated MCF-7 cells. These studies show that DAL-1/4.1B induces apoptosis via caspase 8 activation and that hypomethylation of cellular proteins increases apoptosis as well as DAL-1/4.1B protein levels. These findings suggest that the interaction of the tumor suppressor DAL-1/4.1B and protein methylation pathway components is biologically important in controlling tumorigenesis.

Results

DAL-1/4.1B induces apoptosis in MCF-7 cells via a caspase 8 dependent pathway

Previous work from this laboratory identified DAL-1/4.1B protein as a growth suppressor and apoptosis-inducing protein in MCF-7 cells, which themselves do not express endogenous DAL-1/4.1B [3]. In agreement with this finding, DAL-1/4.1B-inducible MCF-7 Cl27 cells underwent apoptosis when treated with 2 μM Muristerone A for 48 hours to induce DAL-1/4.1B expression. The presence of DAL-1/4.1B protein was confirmed by both Western blot analysis and flow cytometry (FACS)(Figure 1A and 1B). TUNEL analysis revealed that 48 hours of DAL-1/4.1B protein expression induced apoptosis. Not all cells in the MCF-7 Cl27 clone express robust levels of DAL-1/4.1B protein, even after repeated subcloning. Therefore we also analyzed the sub-population of cells that showed high levels of DAL-1/4.1B protein. In that analysis, apoptosis levels reached approximately 80% (Figure 1C).
Figure 1

Induction of DAL-1/4.1B-expression in MCF7 Cl27 cells induces apoptosis. A. Western blot analysis showing the induction of DAL-1/4.1B protein in MCF-7 Cl27 cells following treatment with 2 μM muristerone for 48 hours. B. Flow cytometric analysis confirming the induction of DAL-1/4.1B in cells treated with 2 μM Muristerone (clear peak) as compared to untreated cells (grey peak). C. The level of apoptosis in the total cell population and in cells specifically expressing measurable levels of DAL-1/4.1B protein was determined by TUNEL assay and detected by FACS.

To better understand the apoptotic mechanisms invoked in MCF-7 cells upon expression of DAL-1/4.1B, global as well as specific caspase activation was examined. FAM-VAD-FMK, a potent inhibitor of caspase activity that irreversibly binds to the reactive cysteine residue of the large subunit of caspases 1–9, was incubated with MCF-7 Cl27 cells with or without induction of DAL-1/4.1B protein expression to assess global caspase activation. These probes utilize carboxyfluorescein(FAM)-labeled peptide fluoromethyl ketone (FMK) caspase inhibitors (FAM-peptide-FMK) and allow the fluorescent detection of active caspases in living cell systems. As shown in Figure 2A, the presence of DAL-1/4.1B protein increased global caspase activation levels by 2.5-fold suggesting that DAL-1/4.1B-induced apoptosis proceeds through a caspase-dependent pathway.
Figure 2

Caspase activation in DAL-1/4.1B-induced MCF-7 cells. A. Global caspase activation in cells treated with 2 μM Muristerone for 48 hours or 1 μM staurosporine (STS) for 4 hours. B. FACS analysis of the activation of Caspases 1, 2, 6, 8, 9, 10 and 13 in cells with and without the induction of DAL-1/4.1B expression by treatment with 2 μM Muristerone for 48 hours. Clear bars represent untreated cells; grey bars represent DAL-1/4.1B-expressing cells. Caspase 1, p = 0.232; Caspase 2, p = 0.64; Caspase 6, p = 0.625; Caspase 8, p = 0.007; Caspase 9, p = 0.16; Caspase 10, p = 0.10; Caspase 13, p = 0.039. C. Caspase 7 activation was investigated by Western blot analysis of PARP cleavage on proten lysates from uninduced and DAL-1/4.1B-induced MCF-7 Cl27 cells. D. Inhibition of caspase 8 activation blocks DAL-1/4.1B-associated apoptosis in a dose-dependent manner. This suggests that caspase 8 activation is critical for the induction of cell death by this tumor suppressor protein.

Three main effector caspases, caspases 3, 6 and 7, are thought to be directly involved in the execution of caspase-dependent apoptosis. Importantly, MCF-7, as well as the derivative DAL-1/4.1B-inducible Cl27 cell line, are Caspase 3-deficient [21]. Therefore, we tested the ability of DAL-1/4.1B expression to activate specific caspases other than caspase 3 and including caspases 1, 2, 6, 7, 8, 9, 10 and 13. Using caspase specific binding peptides, only caspase 8 (p = 0.007) showed highly statistically significant activation when compared with cells without DAL-1/4.B (Figure 2B). Caspase 7 is thought to function downstream of caspase 3 but in some cases, caspase-7 can be activated in caspase-3 deficient cells, inducing cleavage of PARP [2225]. Western blot analysis shows no PARP cleavage in response to induced DAL-1/4.1B expression (Figure 2C), suggesting that Caspase-7 is not specifically activated during DAL-1/4.1B-associated apoptosis in these cells.

If caspase 8 is directly involved in DAL-1/4.1B-associated apoptosis, then inhibition of caspase 8 activation should prevent cells from dying in response to the presence of the DAL-1/4.1B protein. In support of this hypothesis, incubation of DAL-1/4.1B-expressing MCF-7 Cl27 cells with the caspase 8-specific inhibitor z-IETD-FMK resulted in blockage of apoptosis in a dose-dependent manner (Figure 2D). Incubation of cells with this inhibitor in the absence of DAL-1/4.1B protein had no effect. Several publications have previously documented the ability of caspase 8 activation to mediate the cleavage of downstream substrates and induce apoptosis in the absence of activation of downstream effecter caspases 3, 6 and 7 [26]. Therefore, DAL-1/4.1B-induced apoptosis in MCF-7 Cl27 cells may involve a caspase 8-dependent pathway which functions independent of the major effector caspase pathways.

While our data suggests that caspase 8 is primarily involved in DAL-1/4.1B mediated apoptosis in MCF-7 cells, the possibility that caspase 3 would also be activated, if present, was tested. To this end, caspase 3-expressing MCF-7 Cl27 cells were generated [21, 27] and caspase 3 expression confirmed in several isolated clones (Figure 3A). Subsequent induction of DAL-1/4.1B expression in these clones (as represented by Cl27.11 in Figure 3B) did not enhance the previously measured level of apoptosis in these cells (8%) [3] suggesting that DAL-1/4.1B-associated apoptosis does not require this major effector caspase.
Figure 3

Restoration of caspase 3 expression does not enhance DAL-1/4.1B-induced apoptosis. A. Western blot analysis of Caspase-3-deficient MCF-7 and DAL-1/4.1B-inducible Cl27 cells. CL 27.2, CL 27.7 and CL 27.11 are Caspase-3-expressing MCF-7 DAL-1/4.1B-inducible clones. B. Apoptosis levels measured up to 4 days after DAL-1/4.1B -expression in MCF-7 CL 27.11 cells. TUNEL positive cells were counted in a microscopic field of 200 cells as previously reported [3] The expression of Caspase-3 does not significantly alter the apoptosis levels over that previously shown in Figure 2A for the caspase-3-deficient MCF-7 Cl27 cells.

Protein methylation and DAL-1/4.1B cooperate to induce apoptosis in MCF-7 cells

Given that DAL-1/4.1B has recently been shown to modulate the ability of the arginine methyltransferases PRMT3 [12] and PRMT5 [20] to methylate cellular substrates, we asked whether posttranslational protein methylation might also play a role in DAL-1/4.1B-associated apoptosis. To address this, DAL-1/4.1B-inducible MCF-7 Cl27 cells were grown for 48 hours in the presence of 30 μM periodate-oxidized adenosine (AdOX). AdOX, an inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase, which elevates the levels of AdoHcy in cultured mammalian cells. Since AdoHcy is a product inhibitor of methyltransferases using AdoMet as the methyl donor, AdOX can reduce the activity of protein methyltransferases in cultured cells and consequently allow the accumulation of hypomethylated protein substrates. Hypomethylated MCF-7 Cl27 cell lysates were collected and the methylation status of endogenous PRMT substrates analyzed by Western blot using the anti-asymmetric dimethylarginine antibody ASYM 24. Figure 4A shows that asymmetrically dimethylated proteins in MCF-7 Cl27 cells were significantly, although not completely, inhibited by treatment with 30 μM AdOX. Higher AdOX concentrations resulted in significant cellular toxicity (data not shown).
Figure 4

Hypomethylation modulates apoptosis in MCF-7 Cl27 cells. A. Western blot analysis with the anti-asymmetric dimethylarginine antibody ASYM24 (Upstate Biochemical) shows significant reduction in the methylation of proteins in MCF-7 Cl27 cells when treated with 30 μM AdOX for 48 hours. B. TUNEL and global caspase activation assays show that AdOX-associated hypomethylation specifically increases the percentage of apoptotic cells in the presence of DAL-1/4.1B protein. Grey bars show the percentage of apoptotic cells as measured by TUNEL. Black bars represent the percentage of global caspase activation.

To determine the effect of protein hypomethylation on DAL-1/4.1B-induced apoptosis, MCF-7 Cl27 cells were induced to express DAL-1/4.1B protein in the presence of 30 μM AdOX and analyzed for apoptosis levels as well as global caspase activation. While treatment with AdOX had no effect on apoptosis, protein hypomethylation significantly increases the induction of cell death by DAL-1/4.1B (Figure 4B). This suggests that the modulation of protein methylation may be an important mechanism of DAL-1/4.1B-induced apoptosis.

Discussion

Apoptosis is traditionally characterized by a series of morphological features such as chromatin condensation, nuclear fragmentation, and the appearance of membrane-enclosed apoptotic bodies. Many proteins, including the caspase family of aspartate-specific cysteine proteases, have been reported to play a pivotal role in the apoptotic process [2831]. However, caspase-independent pathways are emerging [32]. It has been shown previously that expression of the tumor suppressor DAL-1/4.1B can induce apoptosis in MCF-7 breast cancer cells [3] but the mechanism(s) involved have not yet been identified. More recently, it was reported that DAL-1/4.1B interacts with members of the protein arginine N-methyltransferase family (PRMTs) and modulates the posttranslational methylation of cellular substrates. In addition, it was determined that DAL-1/4.1B was not itself a substrate for this post-translational arginine methylation [12]. In this report, we examined the caspase-dependence of DAL-1/4.1B-induced apoptosis and the effect of inhibiting protein methylation on this cell death in MCF-7 cells, to determine if post-translational protein methylation is one potential mechanism through which DAL-1/4.1B exerts its growth suppressive properties.

Previously [3] and in this report, induction of DAL-1/4.1B expression was shown to induce apoptosis in MCF-7 cells. Examination of a series of caspases revealed that these apoptotic events occurred without activation of the three major effector caspases (caspases 3, 6 and 7) but did result in a significant increase in caspase 8 (p = 0.007) activation. The addition of the caspase 8-specific inhibitor z-VAD-FMK blocked the ability of DAL-1/4.1B to stimulate apoptosis in these cells in a dose dependent manner. Furthermore, restoration of caspase 3 expression did not increase the measured levels of apoptosis following DAL-1/4.1B expression demonstrating that this caspase is not activated even when present. However, restoration of caspase-3 expression was recently shown to sensitize MCF-7 cells to radiation-induced apoptosis exhibiting the hallmarks of a traditional effector caspase activation pathway [33]. Our results suggest that cell death induced by DAL-1/4.1B expression does not proceed via a classic caspase activation cascade, but rather relies solely on one caspase, caspase 8.

This lack of effector caspase activation in DAL-1/4.1B-induced apoptosis is intriguing. Activation of these caspases is one of the major characteristics in the programmed cell death process [34]. However, some cells survive caspase activation, and accumulating evidence suggests that many caspase-activating apoptotic stimuli, including oncogenes, p53, DNA-damaging drugs, proapoptotic Bcl2- family members, cytotoxic lymphocytes, and in some cases even death receptors, do not necessarily require activation of the known effector caspases for programmed cell death to occur [35].

Activation of upstream caspases such as caspase 8 has been reported in both mitochondrial-independent and -dependent pathways [3639]. Activated caspase 8 has been reported to trigger cell death by activating either effector caspases 3, 6 or 7, or DNA damage enzymes such as endonuclease G and AIF. Caspase-induced release of EndoG from mitochondria results in cell apoptosis. Li and colleagues [26] reported apoptosis with activated caspase 8 without activation of effector caspases. A parallel condition – activated caspase 8 without activation of caspases 3, 6, or 7 – occurs when DAL-1/4.1B protein is expressed in MCF-7 cells although preliminary studies did not show the release of EndoG into the cytoplasm (data not shown).

Caspase-8 can also cross talk with calpain-dependent apoptotic pathways. Benjamin and colleagues found that both caspase-8 inhibitor z-IETD and calpain inhibitors can protect mature mouse oligodendrocytes from cell death initiated by staurosporine, thapsigargin and kainite [40]. Their results suggest that crosstalk occurs between the caspase and calpain pathways, upstream of an irreversible cascade leading to cell death in mature oligodendrocytes. The cross-talk between caspase 8 and calpain has also been identified in vascular smooth muscle cells during Fas-associated apoptosis [41]. DAL-1/4.1B could also induce apoptosis through membrane protein proteases such as calpains. A related 4.1 family tumor suppressor, NF2, has been shown to interact with calpains in neurofibromatosis-related tumors [42]. In the case of the NF2 tumors, some patients lacking functional mutations in the NF2 gene have concomitant overactive calpain protease activity, which effectively degrades the existing NF2 protein, creating a "loss of tumor suppressor protein"-equivalent environment. The potential relationship between DAL-1/4.1B and calpains and their relationship to apoptosis is important to examine further.

Although protein methylation has been shown to be involved in such cellular processes as signal transduction and transcription [43] no evidence connecting protein methylation and apoptosis has been reported previously. AdOX, an inhibitor of s-adenosylhomocysteine (AdoHcy) hydrolase, can inhibit methylation by elevating the cellular level of AdoHcy to inhibit the activity of methyltransferases [44]. As AdoHcy is a general inhibitor of the once carbon metabolism pathway, its elevation could also inhibit DNA as well as RNA methylation events. Hypomethylation of cellular methyl-accepting protein substrates by AdOX has been demonstrated previously [4449] and confirmed in this study (Figure 4A). Rat pheochromocytoma (PC12) cells treated with 30 μM AdOX for 72 hours were found to undergo a 50% decrease in growth rate [44]. In the present analysis, apoptosis levels in MCF-7 cells were not affected by the hypomethylating treatment of cells with 30 μM AdOX for 48 hours. However, AdOX treatment appeared to enhance apoptosis when the DAL-1/4.1B protein was expressed in MCF-7 cells. RNA methylation has also been shown to be altered by AdOX treatment [50] and such methylation could regulate protein synthesis [5153]. DAL-1/4.1B has previously been shown not to be a substrate for PRMT3- (Singh et al., 2004) or PRMT5-mediated (Jiang et al., 2005) arginine methylation but no information is currently available as to the presence of methylated DAL-1/4.1B mRNA species.

Further investigation into the relationship between DAL-1/4.1B, protein methylation and apoptosis is required to determine the exact mechanism(s) by which tumor cell growth and apoptosis are regulated by these proteins. The determination that caspase 8 activation occurs in the absence of effector caspase activation suggests a potentially novel pathway combining aspects of both traditional caspase-dependent cell death and the emerging effector caspase-independent pathways of apoptosis. Furthermore, the interaction between a tumor suppressor (DAL-1/4.1B) and a post-translational methylation enzyme (PRMTs) is likely to be an important modulator of this pathway and so be of significant biological importance in controlling tumorigenesis in breast cancer cells.

Conclusion

In this report, caspase 8-specific activation by the tumor suppressor DAL-1/4.1B is identified in the absence of activation of effector caspases 3, 6, or 7, suggesting a potentially novel apoptotic pathway combining aspects of both traditional caspase-dependent cell death and the emerging effector caspase-independent pathways of apoptosis. Second it is shown that there is cooperation between DAL-1/4.1B and post-translational protein methylation in the induction of apoptosis in MCF-7 cells. This suggests that the previously published interaction between the tumor suppressor DAL-1/4.1B and the post-translational methylation enzymes (PRMTs) is likely to be an important modulator of this apoptotic pathway and so be of significant biological importance in controlling tumorigenesis in breast cancer cells.

Methods

Cell culture

MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in MEM with 10% Fetal Calf Serum (FCS), sodium pyruvate, non-essential amino acids and insulin. The MCF-7 Cl27 cell line is a DAL/4.1B-inducible cell line generated from the parental MCF-7 cell line using the Ecdysone Muristerone-inducible Expression Kit (InVitrogen) [3]. DAL-1/4.1B expression is induced by the addition of 2 μM Muristerone to the culture medium for 48 hours. Hypomethylation of cells was carried out by addition of 30 μM AdOX (adenosine, periodate oxidized; Sigma) to the culture medium for 48 hours. A positive control for apoptotic cells was obtained by incubating cells in 1 μM Staurosporine (STS, Sigma) for 4 hours.

Western blot

Cell lysates were prepared in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) with protease inhibitors (Roche). Electrophoresis was performed on 10% SDS-PAGE gel (BioRad) and transferred onto PVDF plus membrane (MSI Inc) using the BioRad mini Protean II transfer system as previously described [1]. PARP antibody (Cell Signaling) was used at a 1:1000 dilution for the assessment of caspase 7 activation. Inhibition of protein methylation by AdOX treatment was determined using the anti-asymmetric arginine methylation antibody ASYM24 (Upstate Biotech) at a 1:1000 dilution. Detection was performed using the ECL Plus Western Detection Reagents (Amersham).

FACS analysis

MCF-7 Cl27 cells were treated with 2 μM muristerone A with or without the addition of 30 μM AdOX for 48 hours prior to FACS analysis. For DAL-1/4.1B protein level determinations, cells were collected following trypsinization with 10 mM EDTA/1× PBS followed by two 1× washes in PBS. All procedures were performed on ice. Resuspended cells were then washed twice in 1× Staining buffer (1× PBS containing 2% FCS, 1% human serum, 10 mM HEPES, and 0.025% sodium azide) and collected by centrifugation at 200 g for 5 minutes at 4°C. Cells were then fixed in 0.25% paraformaldehyde in 1× staining buffer on ice for 30 minutes to 60 minutes. The buffer was then replaced and cells permeablized in 0.2% Tween-20/1XPBS at 37°C for 15 minutes after which 50 μl of human serum was added to resuspend the cells. Primary antibody (50 μl, rabbit polyclonal anti-DAL-1/4.1B antibody diluted 1:50) was added and incubated on ice for 30 minutes. Cells were then washed twice in 0.2% Tween-20/1XPBS buffer. Secondary antibody (phycoerythrin conjugate anti-rabbit IgG H+L (Vector) was added on ice in the dark for 20 minutes followed by two washes in 0.2% Tween-20/1XPBS buffer. Cells were then analyzed on the FAC Calibur system (Becton Dickinson).

Apoptosis levels were assessed by Annexin-V (BD Bioscience) and TUNEL (Roche) assays following manufacturer's recommendations. Briefly, cells were trypsinized in 10 mM EDTA/1XPBS and approximately 5 × 104 MCF-7 Cl27 cells were cultured in 500 μl medium in a 24 well plate overnight prior to the addition of 2 μM muristerone with or without 30 μM AdOX. Fresh medium was added and cells were grown for 48 hours before measurement of apoptosis levels. For cells treated with staurosporine (STS), approximately 1 × 105 cells were treated with 1 μM STS for 4 hours.

Caspase activation was analyzed using Carboxyfluorescein Caspase Detection Kits – FAM-VAD-FMK #FAM100 (Global Caspase), FAM-YVAD-FMK #FMK 600 (Caspase 1), FAM-VDVAD-FMK #FMK700 (Caspase 2), FAM-VEID-FMK #FAM500 (Caspase 6), FAM-LETD-FMK #FAM300 (Caspase 8), and FAM-LEHD-FMK #FAM400 (Caspase 9) from Cell Technology, and FAM-AEVD-FMK (Caspase 10) and FAM-LEED-FMK (Caspase 13) from Immunochemistry Technology, LLC. APO LOGIX Carboxyfluoroscein Caspase Detection Kits label active caspases in living cells undergoing apoptosis. For caspase activation assays, cells were plated as described above after which 20 ml of 30× caspase solution was added. Plates were incubated at 37°C for 1.5 hours in the dark. The medium of each sample was then collected by centrifugation at 200 g for 10 minutes at 4°C. Remaining attached cells were washed with 1× washing buffer and added to the non-adherent cell pellets. Attached cells were then detached by treatment with 0.01 M EDTA/1× PBS and all cell solutions combined for analysis on the FAC Calibur system.

Caspase 8 inhibition and the effect on apoptosis levels with and without expression of DAL-1/4.1B protein were examined using the caspase 8-specific inhibitor z-IETD-FMK (Calbiochem). MCF7 Cl27-inducible cells were incubated with 0 mM, 15 μM, or 50 μM of inhibitor for one hour prior to the induction of DAL-1/4.1B protein expression and subsequent measurement of apoptosis by Annexin V staining after 48 hours.

List of abbreviations

AdoHcy: 

S-adenosylhomocysteine

AdOX: 

adenosine dialdehyde

DAL-1: 

differentially-expressed in adenocarcinoma of the lung

FACS: 

fluorescence activated cell sorting

FCS: 

Fetal calf serum

FERM: 

4.1/ezrin/radixin/moesin domain

NF2: 

neurofibromatosis 2

PBS: 

phosphate buffered saline

PRMT: 

protein arginine N-methyltransferase

SDS-PAGE: 

sodium dodecyl sulfate polyacrylamide gel electrophoresis

STS: 

staurosporine.

Declarations

Acknowledgements

We are grateful to Dr. Oliver Bögler for his experimental guidance and critical reading of this manuscript. We also thank Dr. Tom Mikkelsen for his support and Drs. Kevin Bobbitt, Cathie Miller, and Chaya Brodie for their help with the FACS analysis and caspase-specific inhibitors. This work was supported by funds from the Hermelin Brain Tumor Center.

Authors’ Affiliations

(1)
Hermlein Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital
(2)
Brain Tumor Center, Department of Neuro-Oncology, M D Anderson Cancer

References

  1. Tran YK, Bogler O, Gorse KM, Wieland I, Green MR, Newsham IF: A novel member of the NF2/ERM/4.1 superfamily with growth suppressing properties in lung cancer. Cancer Res. 1999, 59 (1): 35-43.PubMedGoogle Scholar
  2. Chishti AH, Kim AC, Marfatia SM, Lutchman M, Hanspal M, Jindal H, Liu SC, Low PS, Rouleau GA, Mohandas N, Chasis JA, Conboy JG, Gascard P, Takakuwa Y, Huang SC, Benz EJJ, Bretscher A, Fehon RG, Gusella JF, Ramesh V, Solomon F, Marchesi VT, Tsukita S, Tsukita S, Hoover KB: The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane. Trends Biochem Sci. 1998, 23 (8): 281-282. 10.1016/S0968-0004(98)01237-7View ArticlePubMedGoogle Scholar
  3. Charboneau AL, Singh V, Yu T, Newsham IF: Suppression of growth and increased cellular attachment after expression of DAL-1 in MCF-7 breast cancer cells. Int J Cancer. 2002, 100 (2): 181-188. 10.1002/ijc.10470View ArticlePubMedGoogle Scholar
  4. Gutmann DH, Donahoe J, Perry A, Lemke N, Gorse K, Kittiniyom K, Rempel SA, Gutierrez JA, Newsham IF: Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas. Hum Mol Genet. 2000, 9 (10): 1495-1500. 10.1093/hmg/9.10.1495View ArticlePubMedGoogle Scholar
  5. Friesen WJ, Paushkin S, Wyce A, Massenet S, Pesiridis GS, Van Duyne G, Rappsilber J, Mann M, Dreyfuss G: The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins. Mol Cell Biol. 2001, 21 (24): 8289-8300. 10.1128/MCB.21.24.8289-8300.2001PubMed CentralView ArticlePubMedGoogle Scholar
  6. Marfatia SM, Lue RA, Branton D, Chishti AH: In vitro binding studies suggest a membrane-associated complex between erythroid p55, protein 4.1, and glycophorin C. J Biol Chem. 1994, 269 (12): 8631-8634.PubMedGoogle Scholar
  7. Tang CJ, Tang TK: The 30-kD domain of protein 4.1 mediates its binding to the carboxyl terminus of pICln, a protein involved in cellular volume regulation. Blood. 1998, 92 (4): 1442-1447.PubMedGoogle Scholar
  8. Morrison H, Sherman LS, Legg J, Banine F, Isacke C, Haipek CA, Gutmann DH, Ponta H, Herrlich P: The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44. Genes Dev. 2001, 15 (8): 968-980. 10.1101/gad.189601PubMed CentralView ArticlePubMedGoogle Scholar
  9. Obremski VJ, Hall AM, Fernandez-Valle C: Merlin, the neurofibromatosis type 2 gene product, and beta1 integrin associate in isolated and differentiating Schwann cells. J Neurobiol. 1998, 37 (4): 487-501. 10.1002/(SICI)1097-4695(199812)37:4<487::AID-NEU1>3.0.CO;2-BView ArticlePubMedGoogle Scholar
  10. Sherman LS, Gutmann DH: Merlin: hanging tumor suppression on the Rac. Trends Cell Biol. 2001, 11 (11): 442-444. 10.1016/S0962-8924(01)02128-6View ArticlePubMedGoogle Scholar
  11. Yu T, Robb VA, Singh V, Gutmann DH, Newsham IF: The 4.1/ezrin/radixin/moesin domain of the DAL-1/Protein 4.1B tumour suppressor interacts with 14-3-3 proteins. Biochem J. 2002, 365 (Pt 3): 783-789.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Singh V, Miranda TB, Jiang W, Frankel A, Roemer ME, Robb VA, Gutmann DH, Herschman HR, Clarke S, Newsham IF: DAL-1/4.1B tumor suppressor interacts with protein arginine N-methyltransferase 3 (PRMT3) and inhibits its ability to methylate substrates in vitro and in vivo. Oncogene. 2004, 23 (47): 7761-7771. 10.1038/sj.onc.1208057View ArticlePubMedGoogle Scholar
  13. Bedford MT, Frankel A, Yaffe MB, Clarke S, Leder P, Richard S: Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. J Biol Chem. 2000, 275 (21): 16030-16036. 10.1074/jbc.M909368199View ArticlePubMedGoogle Scholar
  14. Chen D, Huang SM, Stallcup MR: Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300. J Biol Chem. 2000, 275 (52): 40810-40816. 10.1074/jbc.M005459200View ArticlePubMedGoogle Scholar
  15. Fabbrizio E, El Messaoudi S, Polanowska J, Paul C, Cook JR, Lee JH, Negre V, Rousset M, Pestka S, Le Cam A, Sardet C: Negative regulation of transcription by the type II arginine methyltransferase PRMT5. EMBO Rep. 2002, 3 (7): 641-645. 10.1093/embo-reports/kvf136PubMed CentralView ArticlePubMedGoogle Scholar
  16. Koh SS, Chen D, Lee YH, Stallcup MR: Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem. 2001, 276 (2): 1089-1098. 10.1074/jbc.M004228200View ArticlePubMedGoogle Scholar
  17. Mowen KA, Schurter BT, Fathman JW, David M, Glimcher LH: Arginine methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes. Mol Cell. 2004, 15 (4): 559-571. 10.1016/j.molcel.2004.06.042View ArticlePubMedGoogle Scholar
  18. Mowen KA, Tang J, Zhu W, Schurter BT, Shuai K, Herschman HR, David M: Arginine methylation of STAT1 modulates IFNalpha/beta-induced transcription. Cell. 2001, 104 (5): 731-741. 10.1016/S0092-8674(01)00269-0View ArticlePubMedGoogle Scholar
  19. Wang H, Huang ZQ, Xia L, Feng Q, Erdjument-Bromage H, Strahl BD, Briggs SD, Allis CD, Wong J, Tempst P, Zhang Y: Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science. 2001, 293 (5531): 853-857. 10.1126/science.1060781View ArticlePubMedGoogle Scholar
  20. Jiang W, Roemer ME, Newsham IF: The tumor suppressor DAL-1/4.1B modulates protein arginine N-methyltransferase 5 activity in a substrate-specific manner. Biochem Biophys Res Commun. 2005, 329 (2): 522-530. 10.1016/j.bbrc.2005.01.153View ArticlePubMedGoogle Scholar
  21. Hattangadi DK, DeMasters GA, Walker TD, Jones KR, Di X, Newsham IF, Gewirtz DA: Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell. Biochem Pharmacol. 2004, 68 (9): 1699-1708. 10.1016/j.bcp.2004.06.033View ArticlePubMedGoogle Scholar
  22. Le XF, Marcelli M, McWatters A, Nan B, Mills GB, O'Brian CA, Bast RCJ: Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C alpha activity. Oncogene. 2001, 20 (57): 8258-8269. 10.1038/sj.onc.1205039View ArticlePubMedGoogle Scholar
  23. Lindahl T, Satoh MS, Poirier GG, Klungland A: Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks. Trends Biochem Sci. 1995, 20 (10): 405-411. 10.1016/S0968-0004(00)89089-1View ArticlePubMedGoogle Scholar
  24. Metkar SS, Wang B, Ebbs ML, Kim JH, Lee YJ, Raja SM, Froelich CJ: Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis. J Cell Biol. 2003, 160 (6): 875-885. 10.1083/jcb.200210158PubMed CentralView ArticlePubMedGoogle Scholar
  25. Satoh MS, Lindahl T: Role of poly(ADP-ribose) formation in DNA repair. Nature. 1992, 356 (6367): 356-358. 10.1038/356356a0View ArticlePubMedGoogle Scholar
  26. Li LY, Luo X, Wang X: Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 2001, 412 (6842): 95-99. 10.1038/35083620View ArticlePubMedGoogle Scholar
  27. Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM: Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995, 81 (5): 801-809. 10.1016/0092-8674(95)90541-3View ArticlePubMedGoogle Scholar
  28. Ashkenazi A, Dixit VM: Apoptosis control by death and decoy receptors. Curr Opin Cell Biol. 1999, 11 (2): 255-260. 10.1016/S0955-0674(99)80034-9View ArticlePubMedGoogle Scholar
  29. Bogler O, Mikkelsen T: Angiogenesis and apoptosis in glioma: Two arenas for promising new therapies. J Cell Biochem. 2005.Google Scholar
  30. Thornberry NA, Lazebnik Y: Caspases: enemies within. Science. 1998, 281 (5381): 1312-1316. 10.1126/science.281.5381.1312View ArticlePubMedGoogle Scholar
  31. Strasser A, O'Connor L, Dixit VM: Apoptosis signaling. Annu Rev Biochem. 2000, 69: 217-245. 10.1146/annurev.biochem.69.1.217View ArticlePubMedGoogle Scholar
  32. Kroemer G, Martin SJ: Caspase-independent cell death. Nat Med. 2005, 11 (7): 725-730. 10.1038/nm1263View ArticlePubMedGoogle Scholar
  33. Yang XH, Edgerton S, Thor AD: Reconstitution of caspase-3 sensitizes MCF-7 breast cancer cells to radiation therapy. Int J Oncol. 2005, 26 (6): 1675-1680.PubMedGoogle Scholar
  34. Shi Y: Caspase activation: revisiting the induced proximity model. Cell. 2004, 117 (7): 855-858. 10.1016/j.cell.2004.06.007View ArticlePubMedGoogle Scholar
  35. Cregan SP, Dawson VL, Slack RS: Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene. 2004, 23 (16): 2785-2796. 10.1038/sj.onc.1207517View ArticlePubMedGoogle Scholar
  36. Clement MV, Hirpara JL, Pervaiz S: Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria. Cell Death Differ. 2003, 10 (11): 1273-1285. 10.1038/sj.cdd.4401302View ArticlePubMedGoogle Scholar
  37. Morishima N: Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action. Genes Cells. 1999, 4 (7): 401-414. 10.1046/j.1365-2443.1999.00270.xView ArticlePubMedGoogle Scholar
  38. Ruiz-Ruiz C, Lopez-Rivas A: Mitochondria-dependent and -independent mechanisms in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis are both regulated by interferon-gamma in human breast tumour cells. Biochem J. 2002, 365 (Pt 3): 825-832.PubMed CentralView ArticlePubMedGoogle Scholar
  39. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME: Two CD95 (APO-1/Fas) signaling pathways. Embo J. 1998, 17 (6): 1675-1687. 10.1093/emboj/17.6.1675PubMed CentralView ArticlePubMedGoogle Scholar
  40. Benjamins JA, Nedelkoska L, George EB: Protection of mature oligodendrocytes by inhibitors of caspases and calpains. Neurochem Res. 2003, 28 (1): 143-152. 10.1023/A:1021612615554View ArticlePubMedGoogle Scholar
  41. Schaub FJ, Liles WC, Ferri N, Sayson K, Seifert RA, Bowen-Pope DF: Fas and Fas-associated death domain protein regulate monocyte chemoattractant protein-1 expression by human smooth muscle cells through caspase- and calpain-dependent release of interleukin-1alpha. Circ Res. 2003, 93 (6): 515-522. 10.1161/01.RES.0000093205.42313.7CView ArticlePubMedGoogle Scholar
  42. Kimura Y, Koga H, Araki N, Mugita N, Fujita N, Takeshima H, Nishi T, Yamashima T, Saido TC, Yamasaki T, Moritake K, Saya H, Nakao M: The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas. Nat Med. 1998, 4 (8): 915-922. 10.1038/nm0898-915View ArticlePubMedGoogle Scholar
  43. McBride AE, Silver PA: State of the arg: protein methylation at arginine comes of age. Cell. 2001, 106 (1): 5-8. 10.1016/S0092-8674(01)00423-8View ArticlePubMedGoogle Scholar
  44. Najbauer J, Aswad DW: Diversity of methyl acceptor proteins in rat pheochromocytoma (PC12) cells revealed after treatment with adenosine dialdehyde. J Biol Chem. 1990, 265 (21): 12717-12721.PubMedGoogle Scholar
  45. Johnson BA, Najbauer J, Aswad DW: Accumulation of substrates for protein L-isoaspartyl methyltransferase in adenosine dialdehyde-treated PC12 cells. J Biol Chem. 1993, 268 (9): 6174-6181.PubMedGoogle Scholar
  46. Najbauer J, Johnson BA, Aswad DW: Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells. Anal Biochem. 1991, 197 (2): 412-420. 10.1016/0003-2697(91)90413-NView ArticlePubMedGoogle Scholar
  47. Najbauer J, Johnson BA, Aswad DW: Analysis of stable protein methylation in cultured cells. Arch Biochem Biophys. 1992, 293 (1): 85-92. 10.1016/0003-9861(92)90369-8View ArticlePubMedGoogle Scholar
  48. Najbauer J, Johnson BA, Young AL, Aswad DW: Peptides with sequences similar to glycine, arginine-rich motifs in proteins interacting with RNA are efficiently recognized by methyltransferase(s) modifying arginine in numerous proteins. J Biol Chem. 1993, 268 (14): 10501-10509.PubMedGoogle Scholar
  49. Najbauer J, Orpiszewski J, Aswad DW: Molecular aging of tubulin: accumulation of isoaspartyl sites in vitro and in vivo. Biochemistry. 1996, 35 (16): 5183-5190. 10.1021/bi953063gView ArticlePubMedGoogle Scholar
  50. Hermes M, Osswald H, Mattar J, Kloor D: Influence of an altered methylation potential on mRNA methylation and gene expression in HepG2 cells. Exp Cell Res. 2004, 294 (2): 325-334. 10.1016/j.yexcr.2003.12.001View ArticlePubMedGoogle Scholar
  51. Glazer RI, Knode MC: Neplanocin A. A cyclopentenyl analog of adenosine with specificity for inhibiting RNA methylation. J Biol Chem. 1984, 259 (21): 12964-12969.PubMedGoogle Scholar
  52. Jacquemont B, Huppert J: Inhibition of viral RNA methylation in herpes simplex virus type 1-infected cells by 5' S-isobutyl-adenosine. J Virol. 1977, 22 (1): 160-167.PubMed CentralPubMedGoogle Scholar
  53. Mertens PP, Payne CC: The effects of S-adenosyl methionine (AdoMet) and its analogues on the control of transcription and translation in vitro of the mRNA products of two cytoplasmic polyhedrosis viruses. Virology. 1983, 131 (1): 18-29. 10.1016/0042-6822(83)90529-9View ArticlePubMedGoogle Scholar

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