Protein Expression Profiles of Necrosis and Apoptosis Induced by 5-Fluoro-2′-deoxyuridine in Mouse Cancer Cells Akira Sato, Akito Satake, Akiko Hiramoto, Yusuke Wataya, and Hye-Sook Kim* Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan Received November 17, 2009
We have investigated the molecular mechanisms regulating the necrosis and apoptosis that occur on treatment of mouse mammary tumor FM3A cells with 5-fluoro-2′-deoxyuridine (FUdR), a potent anticancer agent, using the original clone F28-7 and its variant F28-7-A cells. Previously, we reported an interesting observation that FUdR induces a necrotic morphology in F28-7 but an apoptotic morphology in F28-7-A cells. We have now analyzed the protein expression profiles of these FUdRinduced necrosis and apoptosis. Thus, proteome analysis of these clones by two-dimensional gel electrophoresis and mass spectrometry showed that the cytoplasmic intermediate filament protein, cytokeratin-19, is expressed at a significantly higher level in F28-7 than in F28-7-A cells. This strong expression was detected both in untreated and FUdR-treated stages of F28-7 cells. We interpreted this phenomenon as suggesting that cytokeratin-19 possesses a function in leading the cell to apoptosis. We performed a knockdown of cytokeratin-19 expression in F28-7 cells by use of the small interfering RNA technique. Indeed, a lowering of the cytokeratin-19 expression down to the level in F28-7-A occurred, and the FUdR-induced death morphology of this knockdown F28-7 was apoptosis, instead of the necrosis usually observable in the FUdR-treated F28-7. It is known that the cytoskeletal protein cytokeratin-19 undergoes caspase-mediated degradation during apoptosis. Our present finding provides an interesting possibility that cytokeratin-19 may have a key role in regulating cell-death morphology. Keywords: apoptosis • cell death • cytokeratin-19 • 5-fluoro-2′-deoxyuridine (FUdR) • intermediate filament • lamin B1 • necrosis • proteome analysis • siRNA
Introduction Transcriptome and proteome analyses have been performed extensively to identify candidate genes and proteins involved in biological processes. A technique well suited for the analysis of protein compositions, modifications, and translocations is proteome analysis based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry.1 In cancer research, the subject of cell death is important for achieving therapeutic induction of cancer-cell-selective death. Among these two major forms of cell death, apoptosis and necrosis, apoptosis has received a proportionately greater degree of attention than has necrosis.2,3 Proteome analysis has been used extensively to study apoptosis in cells, and more than 100 proteins have been identified as participants in the apoptosis.4 However, this approach has been seldom applied for studies of necrosis. We have investigated the molecular mechanisms regulating necrosis and apoptosis that occur on treatment of mouse mammary tumor FM3A cells with 5-fluoro-2′-deoxyuridine (FUdR), using the original clone F28-7 and its variant F28-7-A cells.5,6 FUdR, a potent anticancer agent, exerts its effect by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation (see ref 7 and references * To whom correspondence should be addressed. Corresponding author: Dr. Hye-Sook Kim, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan. Tel., +81-86-251-7975; Fax, +81-86251-7974; E-mail,
[email protected]. 10.1021/pr9010537
2010 American Chemical Society
therein). Previously, we reported that the treatment can induce in F28-7 cells a breakdown of DNA into chromosome-sized fragments leading to necrosis, and that it can induce in F287-A more extensive DNA cleavage into oligonucleosome-sized fragments and subsequent development of apoptosis.5 Through our recent studies on the gene expression profiles during the cell-death induced by FUdR, we proposed possible mechanisms associated with the necrosis and apoptosis.6 We have aimed at gaining more comprehensive insights into the cellular mechanisms activated during necrosis and apoptosis. The proteome of a cell is highly dependent on the conditions to which the cell is exposed and may respond in a quite complex manner. Current development of proteomics has now enabled us to analyze the protein expression profiles of necrosis induced by FUdR in F28-7 cells and those of apoptosis in F28-7-A cells. Here, we describe the patterns of differentially expressed proteins between these cells, as revealed by the proteome analysis. Also, phenotypic screening by use of small interfering RNAs (siRNAs) led to detection of a number of differentially expressed proteins in these cells. Using this approach, we identified two new regulators of the cell death: the nuclear inner membrane protein lamin B18 and the cytoplasmic intermediate filament-protein cytokeratin-19 (this report). A knockdown of cytokeratin-19 expression in F28-7 cells was performed by use of the siRNA technique, resulting in a decreased expression of cytokeratin-19 down to the level Journal of Proteome Research 2010, 9, 2329–2338 2329 Published on Web 02/15/2010
research articles in F28-7-A which is prone to apoptotic death. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, clearly different from the necrosis that occurs in the FUdR-treated original F28-7.
Materials and Methods Reagents, Antibodies, Cell, and Cell Culture. 5-Fluoro-2′deoxyuridine (FUdR) was obtained from Sigma. FUdR was stored as 2 mM stocks in HPLC-grade water at -20 °C. 4′,6Diamidino-2-phenylindole dihydrochloride (DAPI) was from Invitrogen. A set of four siRNAs against cytokeratin-19 mRNA was used: Mm_Krt1-19_1 FlexiTube siRNA, Catalog number SIO1085735; Mm_Krt1-19_2 FlexiTube siRNA, Catalog number SIO1085742; Mm_Krt1-19_3 FlexiTube siRNA, Catalog number SIO1085749; and Mm_Krt1-19_4 FlexiTube siRNA, Catalog number SIO1085756. AllStars Negative Control siRNA, Catalog number 1027280, was used as a nonsilencing siRNA. These siRNAs were obtained from Qiagen. The primary antibodies, rabbit polyclonal antihuman keratin-19 (K19) and rabbit polyclonal antikeratin-8 antibody, were from ANASPEC. Rabbit polyclonal anti-Annexin-A1 antibody and rabbit polyclonal antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody were from Abcam and Trevigen, respectively. The secondary antibodies; antimouse IgG horseradish peroxidase-linked whole antibody and antirabbit IgG horseradish peroxidase-linked whole antibody were from GE Healthcare. Original-type F28-7 clone and variant F28-7-A clone of mouse mammary tumor FM3A cells used in the study have been described previously.5 The cells were cultured in ES medium (Nissui Pharmaceuticals), supplemented with 2% fetal bovine serum (Gibco) and 0.03% L-glutamine (Wako), in a humidified atmosphere with 5% CO2 at 37 °C. Under these conditions, the doubling time of both F28-7 and F28-7-A cells was approximately 12 h. F28-7 and F28-7-A cells (approximately 5 × 105 cells/mL) were treated with 1 µM FUdR. Cell viability was estimated with a hemocytometer by means of trypan blue-exclusion. Two-Dimensional Gel Electrophoresis (2-DE). Cells were cultured, collected, and washed in ice-cold phosphate-buffered saline (PBS) and then lysed in 1.5 mL of Ready Prep Rehydration/Sample buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.001% bromophenol blue, and 0.2% w/v Bio-Lyte 3/10 ampholytes) prepared according to the manufacturer’s instructions (BioRad), with supplemental addition of Protease-Inhibitor Cocktail for Mammalian Cell (Sigma). The cells were sonicated on ice using Branson sonifier 250, then the lysate was left at room temperature for 30 min. The cell lysate was centrifuged at 10 000× g for 15 min at room temperature and the supernatant containing the solubilized proteins was used directly or stored at -80 °C prior to use. Protein concentrations were determined using the Lowry-method-oriented RCDC protein assay reagent (Bio-Rad). Protein samples from at least three independent experiments were collected for 2-DE analysis. The proteins were separated by large 2-DE (gel size 18 cm × 20 cm). Briefly, samples containing 200 µg of protein in 350 µL Rehydration/ Sample buffer were loaded on the immobilized pH gradient (IPG) strips (ReadyStrips IPG Strips, 17 cm, pH range 3-10 nonlinear: Bio-Rad). Active rehydration and isoelectric focusing (IEF) were performed at 20 °C using PROTEAN IEF Cell (BioRad). After active rehydration (50 V) for at least 12 h, IEF was performed in three steps, that is, conditioning (15 min at 250 V), voltage ramping (250-10,000 V in 2 h), and focusing (5 h 2330
Journal of Proteome Research • Vol. 9, No. 5, 2010
Sato et al. at 10 000 V). The current was set at 95%) ion score were accepted. In the PMF and MS/MS ion search, the significance threshold for positive identification was determined by the Mascot Search program. For HPLC-Chip/MS analysis, database searches were performed against the Swiss-Prot database (release 57.12 of 15Dec-2009; 513 877 sequences; 180 750 753 residues) using a
spectrum Mill MS proteomic workbench offered by Agilent (Software version Rev A. 03. 03. 084. SR4). The search parameters were set so that they allowed the peptide mass tolerance at (2.5 Da, and the fragment ion tolerance at (0.7 Da. The allowance also included matching peptides containing one miscleavage, selection of species (Mus musculus), fixed modification of carbamidomethylated cysteines, and a variable modification of methionine oxidation. Identification of proteins was validated when at least two peptide sequences matched with the database sequences, with concomitant occurrence of the peptide score greater than 11. All MS and MS/MS spectra obtained were searched against the mouse protein database, which was collected from the Swiss-Prot (Mus musculus, 16 214 sequences) database. The molecular masses and isoelectric points were calculated by employing the software Compute pI/MW (www.expasy.ch/ tool/pi_tool.html). Western Blot Analysis. Cells were washed in ice-cold PBS and then whole cell lysates were prepared using Laemmili sample buffer (Bio-Rad). Proteins (5 × 104 cells per lane) were subsequently fractionated under reducing conditions by 7.5% SDS-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane (Millipore). The membrane was then blocked against nonspecific binding by treatment for 1 h with 5% bovine serum albumin in Trisbuffered saline containing 0.1% Tween 20, and then immunoblotted overnight at 4 °C using the respective primary antibody. Next, the membrane was incubated for 1 h at room temperature with a horseradish peroxidase-conjugated antimouse IgG or antirabbit IgG secondary antibody, and the protein bands were visualized using an ECL plus Western blotting detection system (GE Healthcare). Protein expression Journal of Proteome Research • Vol. 9, No. 5, 2010 2331
research articles
Sato et al.
Table 1. Differential Protein-Spots from F28-7 and F28-7-A spot no.
fold differencea
1 2c
2.4 3.3
P14733 P19001
3 4
5.0 2.0
P10107 P38647
5 6
2.0 2.5
P11679 P19157
7
0.2
P38647
8
0.6
P48678 Q60864 Q9CWJ9
9
0.4
Q9JLJ2
accession no.b
P50431
10
0.6
Q8C1B7 Q9CQM9 Q8BK64
11
0.3
Q9QZH3
12 13
0.6 0.5
O08807 Q60631
14
0.4
Q99LP6
15 16
0.3 0.4
Q99LX0 P50446 Q8CGP5
protein name
biological process (function)
Spots stronger in F28-7 than in F28-7-A Lamin B1 Chromatin modificaton Keratin, type I cytoskeletal 19 Cytoskeleton organization (Cytokeratin-19) Annexin A1 Signal transduction Stress-70 protein, mitochondrial Protein folding (GRP 75) Keratin, type II cytoskeletal 8 Cytoskeleton organization Glutathione S-transferase P1 Glutathione metabolism Spots weaker in F28-7 than in F28-7-A Stress-70 protein, mitochondrial Protein folding (GRP 75) Lamin-A/C Chromatin modificaton Stress-induced phosphoprotein 1 Stress response Bifunctional purine biosynthesis Purine nucleotide biosynthesis protein PURH 4-trimethylaminobutyraldehyde Carnitine metabolism dehydrogenase (Aldehyde dehydrogenase 9A1) Serine hydroxymethyltransferase, Glycine metabolism cytosolic Septin-11 Cell cycle Glutaredoxin-3 Redox homeostasis Activator of 90 kDa heat shock Protein folding protein ATPase homologue 1 (AHA1) Peptidyl-prolyl cis-trans Protein folding isomerase E Peroxiredoxin 4 Redox homeostasis Growth factor receptor-bound Signal transduction protein 2 GrpE protein homologue 1, Protein folding mitochondrial Protein DJ-1 Protein folding Keratin, type II cytoskeletal 6A Cytoskeletone organization Histone H2A type 1-F Chromatin modificaton
a Fold difference (F28-7/F28-7-A cells) > 1.5 or e 0.6. b Swiss-prot primary accession number. indicate the identified peptides by MS/MS experiments in MALDI-TOF and/or nano-LC-MS/MS.
was quantified using VersaDoc imaging system (Bio-Rad). The following antibodies were used: anticytokeratin-19 antibody (1:200), anti-GAPDH antibody (1:10 000), antimouse IgG horseradish peroxidase-linked whole antibody (1:20 000), and antirabbit IgG horseradish peroxidase-linked whole antibody (1:20 000). Transfection. Exponentially growing 2 × 105 cells were suspended in 75 µL siPORT electroporation buffer (Ambion) containing cytokeratin-19 siRNA cocktail or nonsilencing siRNA (final concentration 8 × 10-7 M) and introduced into 0.1 cm gap electroporation cuvette (Bio-Rad). The cytokeratin-19 siRNA cocktail was prepared by mixing Mm_Krt1-19_1, Mm_Krt1-19_2, Mm_Krt1-19_3, and Mm_Krt1-19_4 FlexiTube siRNAs. Cells were then electroporated using the BioRad Gene Pulser Xcell at voltage 0.15 kV, pulse length 1000 µs, and number of pulse 1. After electroporation, cells were plated at 5 × 104 cells/mL in fresh ES medium in tissue culture flasks. Forty-eight hours after the electroporation, cells were used for further experiments. 2332
Journal of Proteome Research • Vol. 9, No. 5, 2010
c
theoretical pI
theoretical MW (Da)
5.11 5.28
66785.60 44541.80
6.97 5.91
38734.30 73528.33
5.70 7.69
54565.31 23609.18
5.91
73528.33
6.54 6.40 6.30
74237.57 62582.11 64217.38
6.63
53514.72
6.47
52584.87
6.24 5.42 5.41
49694.64 37778.38 38117.13
5.41
33448.85
6.67 5.89
31052.52 25238.41
8.58
24307.02
6.32 8.04 11.05
20021.31 59335.12 14161.53
See graphic below. Bold-lettered peptide sequences
Results Protein Expression Analysis of Mouse Mammary Tumor FM3A Cells F28-7 and F28-7-A. Cell lysates were analyzed using 2-DE to detect changes in the proteome of F28-7 and F28-7-A cells. Figure 1A and B show typical two-dimensional gels of F28-7 and F28-7-A cell-lysates. Approximately 1800 protein spots per gel were detected within a pI range of 3-10 and a relative molecular mass range of 10-150 kDa. These data were reproducible in two additional independent experiments using newly cultured cells. With a 1.5-fold cutoff, that is, either >1.5 or 1.5 or e 0.6. b Swiss-prot primary accession number. c See graphic below. Bold-lettered peptide sequences indicate the identified peptides by MS/MS experiments in MALDI-TOF and/or nano-LC-MS/MS. d See graphic below. Bold-lettered peptide sequences indicate the identified peptides by MS/MS experiments in MALDI-TOF and/or nano-LC-MS/MS.
chromatin and cytoskeleton-organization showed differential expressions. These proteins include lamin B1, keratin type I cytoskeletal 19 (cytokeratin-19), keratin type II cuticular Hb6, histone H2B type 3-A, keratin type I cytoskeletal 10 (cytokeratin-10), and keratin type I cytoskeletal 14 (cytokeratin-14), all 2334
Journal of Proteome Research • Vol. 9, No. 5, 2010
of them being those involved in the maintenance of nuclear and cell morphology. Interestingly, lamin B1 and cytokeratin19 were higher (1.7, 5.0, respectively) in F28-7 (necrosis cells), compared to F28-7-A cells (apoptosis cells) in this FUdRtreatment (Table 2 and Figure 3). At the untreated stage also,
Protein Expression Profiles of Necrosis and Apoptosis
research articles
Figure 3. Selected regions of two-dimensional gels illustrating individual protein expression changes following FUdR treatment. Pictures showing enlarged areas for lamin B1 (spot 1 and spot f1), and cytokeratin-19 (spot 2 and spot f2).
Figure 4. Western blot validation of cytokeratin-19 protein expression. Whole cell lysates were prepared from F28-7 and F28-7-A cells in untreated (0 h) and FUdR-treated stages (4, 8, 12, 16 h). Expression of cytokeratin-19 and GAPDH proteins were examined by Western blot analysis. Expression of GAPDH was used as an internal control. The patterns shown are results obtained in one experiment. Two additional experiments gave similar results. Expression of cytokeratin-19 protein is represented by the density of cytokeratin-19/GAPDH protein band relative to the zero-time value with F28-7 cells (b). The values for F28-7-A (O) were null at all data points. Results are averages of three independent experiments with error bars showing the (SD in triplicates.
lamin B1 and cytokeratin-19 were higher (2.5, and 3.3, respectively) in F28-7, compared to F28-7-A (Table 1 and Figure 3). Therefore, we interpreted this as suggesting that lamin B1 and cytokeratin-19 could be regulators in the FUdR-induced necrosis and apoptosis. Modulation of FUdR-Induced Cell Death by Silencing Cytokeratin-19 Expression. As described above, lamin B1 and cytokeratin-19 are strongly expressed in F28-7, as compared with those in F28-7-A, both in untreated- and FUdR-treatedstages. Our previous work showed that the knockdown of lamin B1 by siRNA technique can cause a shift from FUdR-induced necrosis to apoptosis.8 In addition, we previously reported that
the cytokeratin-19 mRNA was strongly expressed in FUdRinduced necrosis, as compared with those in FUdR-induced apoptosis, by using cDNA microarray technology.6 The possibility that cytokeratin-19 may also be associated with these differential patterns of cell death morphology was, therefore, investigated. First, we analyzed the expression levels of cytokeratin-19, using Western blotting. As shown in Figure 4, the cytokeratin19 protein was detected for F28-7 but not for F28-7-A cells. The level of cytokeratin-19 protein was indeed higher in F28-7 than in F28-7-A both before and after the FUdR-treatment. In the F28-7 cells, the expression of cytokeratin-19 protein Journal of Proteome Research • Vol. 9, No. 5, 2010 2335
research articles
Figure 5. Knockdown of cytokeratin-19 by RNA interference. F28-7 cells were transfected with nonsilencing siRNA and with cytokeratin-19 siRNA cocktail. Forty-eight hours after the transfection, the levels of expression of cytokeratin-19 and those of GAPDH, an internal control, were examined by Western blot analysis. These results are representative of three independent experiments.
continued to increase until eight-hours of the FUdR-treatment, then decreased gradually, until it reached near to the zero level at 20 h. Next, to test if a lowering of endogenously expressed cytokeratin-19 in F28-7 cells can modulate FUdR-induced necrosis, we carried out a knockdown of the expression in F28-7 cells by using cytokeratin-19 siRNA. Western blot analysis performed for cell extracts at 48 h after the siRNA-transfection at a greaterthan-80% efficiency indicated that the treatment reduced the cytokeratin-19 protein levels, while the glyceraldehyde-3phosphate dehydrogenase (GAPDH) protein levels as a control showed no change (Figure 5). The level of cytokeratin-19 protein expression in F28-7 became as low as that in F28-7-A cells. Thus, the knockdown efficacy was more than 70% in cytokeratin-19 siRNA cocktail-transfected cells, compared to that in the vehicle- and nonsilencing siRNA-transfected cells. Another control experiment in which a nonsilencing siRNA was administered showed no effect on the expression of cytokeratin-19 or GAPDH. The cell viability at 48 h after the transfection was 95.5 ( 0.9% (n ) 3) with the vehicle, 98.9 ( 0.7% (n ) 3) with the nonsilencing siRNA, and 99.1 ( 0.6% (n ) 3) in the cytokeratin-19 siRNA cocktail-transfected cells. The knockdown of cytokeratin-19 in the F28-7 cells did not change the cell viability. In addition, either the nonsilencing siRNA or the cytokeratin-19 siRNA cocktail alone had no effect on the cell
Sato et al. morphology, that is, the morphology-change required subsequent FUdR-treatment (Figure 6, upper diagram). We explored the morphology in the cytokeratin-19 knockeddown F28-7 cells on treatment with 1 µM FUdR. The necrotic morphology in F28-7 and apoptotic morphology in F28-7-A cells were characteristically observed at 21 h after treatment with FUdR. At the 21 h, the controls given vehicle or nonsilencing siRNA showed the cytoplasmic swelling, a feature of necrosis, and the cytokeratin-19 siRNA cocktail-transfected cells, in contrast, showed a typical apoptotic morphology; the membrane blebbing and the formation of apoptotic bodies (Figure 6, bottom diagram). At this point of treatment, the cell viability was 26.7 ( 1.6% (n ) 3) with the vehicle, 26.6 ( 1.9% (n ) 3) with the nonsilencing siRNA, and 28.0 ( 1.1% (n ) 3) with the cytokeratin-19 siRNA cocktail transfection. These results indicate that the knockdown of cytokeratin-19 caused the cells a shift from necrosis to apoptosis, without changing the viability-levels. We confirmed, by microscopic inspection, that almost all the dying cells underwent apoptotic morphologies after the cytokeratin-19 siRNA cocktail transfection with subsequent FUdR treatment.
Discussion We believe that depending on intracellular environment, for example proteome status, a cell is destined to necrosis or to apoptosis. Our previous reports to sort out candidate regulators for the cell death-pathways from the microarray data, however, have been unsuccessful.6 This time, with the use of proteome analysis, we have shown that at the untreated and FUdR-treated stages the nuclear and the cytoplasmic intermediate filament-proteins, lamin B1 and cytokeratin-19, are higher in F28-7 than in F28-7-A cells. Consequently, we focused on these proteins as candidate cell death-regulators of FUdR-induced necrosis and apoptosis. Thus, in our recent publication,8 we reported that a knockdown of lamin B1 by its siRNA induced a shift from necrosis to apoptosis in F28-7 cells, thereby proposing that lamin B1 could be such a regulator. Lamin B1 is one of the nuclear lamins and a key structural component of the lamina, an intermediate filament meshwork that lies beneath the inner nuclear membrane. It is known that the nuclear lamins play a crucial role in fundamental cellular processes, including nuclear organization, chromatin segregation, DNA
Figure 6. Knockdown of cytokeratin-19 shifts FUdR-induced cell-death morphology from necrosis to apoptosis. Forty-eight hours after transfection with the vehicle, the nonsilencing siRNA, or the cytokeratin-19 siRNA cocktail, the F28-7 cells were treated with or without 1 µM FUdR for 21 h and then stained with DAPI as described under Materials and Methods. Morphological changes were analyzed under a fluorescence microscope at ×400 magnification. Two additional experiments gave similar results. 2336
Journal of Proteome Research • Vol. 9, No. 5, 2010
research articles
Protein Expression Profiles of Necrosis and Apoptosis 9-13
replication, and gene expression. Using the same strategy as with lamin B1, we have shown here that a knockdown of cytokeratin-19 in F28-7 results in a shift from necrosis to apoptosis for the FUdR-induced cell-death, leading us to propose that cytokeratin-19 functions, either directly or indirectly, in regulating the cell death-morphology. Both of those proteins, lamin B1 and cytokeratin-19, are constituents of cellular intermediate filaments. Intermediate filaments, together with actin microfilaments and tublin microtubles, comprise the three major cytoskeletal networks that are found in most eukaryotic cells.14 Keratins, which are the largest intermediate filament protein subgroup, can be divided into acidic type-I (K9-K20) and basic type-II (K1-K8).15 Keratin networks are highly dynamic; keratins are reorganized during cell differentiation, mitosis and apoptosis, and they play important cytoprotective and structural support roles for the cells.16,17 Cytokeratin-19 is a member in the largest cytoplasmic intermediate filament-protein subgroup, constituting a key structural component of the cytoskeletal proteins.18,19 Previous reports have indicated that cytokeratin-19 undergoes caspase-mediated degradation during apoptosis.20-22 However, participation of cytokeratin19 in regulation of necrosis and apoptosis has not been documented. Our data suggest that strong expressions of nuclear and cytoplasmic intermediate filament-proteins, lamin B1 and cytokeratin-19, are important in necrosis, and poor expressions of these proteins lead to apoptosis. In addition, it is known that nuclear and cytoplasmic intermediate filament-proteins (e.g., lamin B1, cytokeratin-8, cytokeratin-18, cytokeratin-19, and vimentin) are caspase substrates, which undergo caspase-mediated cleavage during apoptosis.20-24 It seems that a decrease of these intermediate filament-proteins gives greater flexibility in nucleus and cellstructure, thereby leading to apoptosis. As described above, the proteome analysis revealed differently expressed proteins (see Tables 1 and 2). Most of these proteins are for the first time revealed as being differently expressed in necrosis and apoptosis. These proteins need to be investigated further to determine whether they are directly involved in the FUdR-induced necrosis and apoptosis at all. In our proteome analysis, several proteins (e.g., stress-70 protein, galactokinase, annexin A3 and ATP synthase subunit alpha) were detectable in different spots, most likely as a consequence of post-translational modifications or differential mRNA splicings. Our recent work revealed that a release of cytochrome c from mitochondria into the cytoplasm and nucleus occurs in the apoptosis but not in the necrosis.6 Reports from other laboratories have shown that mitochondria play a role in mediating both necrosis and apoptosis.25-27 Interestingly, a number of the mitochondrial proteins exhibit differential expressions in F28-7 and F28-7-A cells either in the untreated stages or in the FUdR-treated stages. Among these proteins, a member of the molecular chaperone Heat shock protein 70 (HSP70) family, stress-70 protein (GRP75), was found to be present in Spot 4 in Figure 1A and Spot 7 in Figure 1B. It is known that GRP75 receives phosphorylation and/or acetylation as post-translational modifications.28,29 We propose that an expression change, that can involve post-translational modification, of these mitochondria-localized proteins play an important role prior to the release of cytochrome c, a
role decisive in determining the FUdR-induced death morphology. Previously, we demonstrated that an inhibition of HSP90, a well-documented chaperone, causes in F28-7 a shift from necrosis to apoptosis in FUdR-induced cell-killing.6 HSP90 is probably one of key regulators in the necrosis and apoptosis, and it would be interesting to explore the relationship between the presently found activities of cytokeratin-19 and lamin B1, the filament constituents, and the role of the ubiquitous cellcomponent HSP90 in the cell death. Finally, our present work shows that these cell-death models and the approach by proteome analysis coupled with usage of interfering RNAs provide a useful methodology in studying molecular mechanisms of necrosis and apoptosis.
Acknowledgment. We thank Dr. Hikoya Hayatsu (Faculty of Pharmaceutical Sciences, Okayama University) for helpful discussions. This research was partly supported by Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (21790078, A.S.). Supporting Information Available: Supplementary Figure 1 and Tables 1-3. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Klose, J.; Kobalz, U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 1995, 16, 1034–1059. (2) Farber, E. Programmed cell death: necrosis versus apoptosis. Mod. Pathol. 1994, 7, 605–609. (3) Kerr, J. F. Shrinkage necrosis: a distinct mode of cellular death. J. Pathol. 1971, 105, 13–20. (4) Thiede, B.; Rudel, T. Proteome analysis of apoptotic cells. Mass Spectrom. Rev. 2004, 23, 333–349. (5) Kakutani, T.; Ebara, Y.; Kanja, K.; Hidaka, M.; et al. Different modes of cell death induced by 5-fluoro-2′-deoxyuridine in two clones of the mouse mammary tumor FM3A cell line. Biochem. Biophys. Res. Commun. 1998, 247, 773–779. (6) Sato, A.; Hiramoto, A.; Uchikubo, Y.; Miyazaki, E.; et al. Gene expression profiles of necrosis and apoptosis induced by 5-fluoro2′-deoxyuridine. Genomics 2008, 92, 9–17. (7) Yoshioka, A.; Tanaka, S.; Hiraoka, O.; Koyama, Y.; et al. Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridineinduced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death. J. Biol. Chem. 1987, 262, 8235–8241. (8) Sato, A.; Hiramoto, A.; Satake, A.; Miyazaki, E.; et al. Association of nuclear membrane protein lamin B1 with necrosis and apoptosis in cell death induced by 5-fluoro-2′-deoxyuridine. Nucleosides Nucleotides Nucleic Acids 2008, 27, 433–438. (9) Cohen, M.; Lee, K. K.; Wilson, K. L.; Gruenbaum, Y. Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. Trends Biochem. Sci. 2001, 26, 41–47. (10) Wilson, K. L.; Zastrow, M. S.; Lee, K. K. Lamins and disease: insights into nuclear infrastructure. Cell 2001, 104, 647–650. (11) Goldman, R. D.; Gruenbaum, Y.; Moir, R. D.; Shumaker, D. K.; et al. Nuclear lamins: building blocks of nuclear architecture. Genes Dev. 2002, 16, 533–547. (12) Burke, B.; Stewart, C. L. Life at the edge: the nuclear envelope and human disease. Nat. Rev. Mol. Cell Biol. 2002, 3, 575–585. (13) Hutchison, C. J. Lamins: building blocks or regulators of gene expression. Nat. Rev. Mol. Cell Biol. 2002, 3, 848–858. (14) Ku, N. O.; Zhou, X.; Toivola, D. M.; Omary, M. B. The cytoskeleton of digestive epithelia in health and disease. Am. J. Physiol. 1999, 277, G1108–1137. (15) Schweizer, J.; Bowden, P. E.; Coulombe, P. A.; Langbein, L.; et al. New consensus nomenclature for mammalian keratins. J. Cell Biol. 2006, 174, 169–174. (16) Omary, M. B.; Coulombe, P. A.; McLean, W. H. Intermediate filament proteins and their associated diseases. N. Engl. J. Med. 2004, 351, 2087–2100.
Journal of Proteome Research • Vol. 9, No. 5, 2010 2337
research articles (17) Omary, M. B.; Ku, N. O.; Tao, G. Z.; Toivola, D. M.; et al. Heads and tails” of intermediate filament phosphorylation: multiple sites and functional insights. Trends Biochem. Sci. 2006, 31, 383–394. (18) Wu, Y. J.; Rheinwald, J. G. A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas. Cell 1981, 25, 627–635. (19) Moll, R.; von Bassewitz, D. B.; Schulz, U.; Franke, W. W. An unusual type of cytokeratin filament in cells of a human cloacogenic carcinoma derived from the anorectal transition zone. Differentiation 1982, 22, 25–40. (20) Ku, N. O.; Liao, J.; Omary, M. B. Apoptosis generates stable fragments of human type I keratins. J. Biol. Chem. 1997, 272, 33197–33203. (21) Ku, N. O.; Omary, M. B. Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation. J. Biol. Chem. 2001, 2276, 26792–26798. (22) Oshima, R. G. Apoptosis and keratin intermediate filaments. Cell Death Differ. 2001, 9, 486–492. (23) Kawahara, A.; Enari, M.; Talanian, R. V.; Wong, W. W.; et al. Fasinduced DNA fragmentation and proteolysis of nuclear proteins. Genes Cells 1998, 3, 297–306.
2338
Journal of Proteome Research • Vol. 9, No. 5, 2010
Sato et al. (24) Byun, Y.; Chen, F.; Chang, R.; Trivedi, M.; et al. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ. 2001, 8, 443–450. (25) Tsujimoto, Y. Cell death regulation by the Bcl-2 protein family in the mitochondria. J. Cell. Physiol. 2003, 195, 158–167. (26) Green, D. R.; Kroemer, G. The pathophysiology of mitochondrial cell death. Science 2004, 305, 626–629. (27) Nakagawa, T.; Shimizu, S.; Watanabe, T.; Yamaguchi, O.; et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 2005, 434, 652–658. (28) Rikova, K.; Guo, A.; Zeng, Q.; Possemato, A.; et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007, 131, 1190–1203. (29) Matsuoka, S.; Ballif, B. A.; Smogorzewska, A.; McDonald, E. R.; et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 2007, 316, 1160–1166.
PR9010537
