Proteomic analysis of ursolic acid-induced apoptosis in cervical carcinoma cells

Cancer Letters 235 (2006) 209–220 www.elsevier.com/locate/canlet

Proteomic analysis of ursolic acid-induced apoptosis in cervical carcinoma cells Eun-Kyoung Yima, Keun-Ho Leeb, Sung-Eun Namkoongb, Soo-Jong Umc,**, Jong-Sup Parka,b,* a

Department of Medical Bioscience, Graduate School of Catholic University, Kangnam St Mary’s Hospital, The Catholic University, 505 Banpo-dong, Seocho-gu, Seoul, 137-040 South Korea b Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Kangnam St Mary’s Hospital, The Catholic University, 505 Banpo-dong, Seocho-gu, Seoul 137-040, South Korea c Department of Bioscience and Biotechnology, Institute of Bioscience, The Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul, South Korea Received 7 June 2004; received in revised form 7 April 2005; accepted 12 April 2005

Abstract Proteomic analyses can efficiently detect the variation of protein in high throughput screening. Using 2DE/MALDI-TOF-MS and SELDI-TOF-MS, we tried to search several cellular proteins that are responsive to ursolic acid (UA) in HeLa cervical carcinoma cells. Compared to control, UA-treated HeLa cells unfolded 25 proteins in significant changes by 2DE/MALDI-TOF-MS, most of which were involved in apoptosis. SELDI-TOF-MS with two types of protein chips profiled and analyzed proteomic features after administration of UA. Interestingly, eight polypeptide peaks can be detected. Further identification and characterization of these proteins may build the molecular basis of UA-induced apoptosis and provide insight into the anti-proliferative mechanism in cervical carcinoma cells. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ursolic acid (UA); Apoptosis; Cervical carcinoma cells; 2DE; MALDI-TOF-MS; SELDI-TOF-MS

1. Introduction

* Corresponding authors. Address: Department of Obstetrics and Gynecology, The Catholic University, 505 Banpo-dong, Seocho-gu, Seoul, South Korea. Tel.: C82 2 590 2596; fax: C82 2 595 8774. ** Tel.: C82 2 3408 3641. E-mail addresses: [email protected] (S.-J. Um), jspark@ catholic.ac.kr (J.-S. Park).

Ursolic acid (UA) (3b-hydroxy-urs-12-en-28-oic acid) is a pentacyclic triterpenoid derived from berries, leaves, flowers, and fruits of medicinal plants; such as Rosemarinus officinalis, Eriobotrya japonica, Calluna vulgaris and Eugenia jumbolana [1]. UA has been shown to suppress tumorigenesis [2], inhibit tumor promotion [3–5] and suppress angiogenesis [6].

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.04.007

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These effects of UA could be mediated through suppression of the expression of lipoxygenase [7], COX-2 [8–10], MMP-9 [11] and iNOS [9]. In addition, UA and its derivatives have been shown to induce apoptosis in a wide variety of cancer cells including breast carcinoma, melanoma, hepatoma, prostate carcinoma, and acute myelogenous leukemia. And these could be explained through inhibition of DNA replication, activation of caspases [12,13], inhibition of protein tyrosine kinases [14], and induction of Ca2C release [15]. Another UA-induced apoptotic mechanism involves in down-regulation of the cellular inhibitor of apoptosis gene [16,17] and inhibition of NF-kB activity [18]. The proteome analysis could discover the qualitative alterations along with the quantitative changes in protein expression level that occurred response to a given set of conditions. The combination of highresolution protein separation by two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) has proven to be an essential proteomics tools to identify differentially expressed proteins and post-translational modifications [19–21]. The application of proteomics technology provides major opportunities to elucidate disease mechanisms and identify new diagnostic markers and therapeutic targets. ProteinChip technology called surface enhanced laser desroption/ ionization-time of flight-mass spectrometry (SELDITOF-MS) has recently been developed to facilitate protein profiling of complex biological materials, e.g. serum, blood, plasma, urine and cell lysates [22–24]. This fast novel technology requires far less material and has a higher reproducibility compared to conventional 1- or 2DE. The search for biomarkers using SELDITOF-MS can directly be used for detecting therapeutic targets for specific medication. This is the first report (describes our initial) to profile differential protein expression in HeLa cervical carcinoma cell lines during UA-induced apoptosis using 2DE/MALDI-TOF-MS and ProteinChip array.

2. Materials and methods 2.1. Cell culture and induction of apoptosis HeLa cells were routinely cultivated in DMEM with 10% FBS at 37 8C in a humidified atmosphere

containing 5% CO2. Ursolic acid (UA) (Sigma, St Louis, MO, USA) was dissolved in DMSO and then diluted 1/1000 with DMSO as the working solution. For induction of apoptosis, HeLa cell was treated with 15-mM UA. UA-treated HeLa cells were harvested by trypsinization after 24-h incubation. 2.2. DAPI staining Treated cells were harvested, washed in 1! PBS, and fixed with 4% paraformaldehyde, stained for 5 min in 0.1 mg of DAPI/ml in a methanol solution, and finally analyzed via fluorescence microscopy to assess chromatin condensation and segregation. 2.3. FACS analysis For cell-cycle analysis, 1!106 cells were cultured in 100 mm dishes. The cells were harvested at the times indicated by trypsin-EDTA digestion. After washing twice with 1! PBS, cells were fixed with ice-cold 100% (vol/vol) ethanol at room temperature for 1 h. The cells were treated with 25 units RNase (Sigma) at room temperature for 30 min and then resuspended in PBS containing 50 mg/ml propidium iodide (PI) (Sigma). DNA fluorescence was measured with a FACS System (Becton-Dickinson Immunocytometer System, San Jose, CA, USA). 2.4. Two-Dimensional electrophoresis (2DE) HeLa cell was treated with 15-mM UA and harvested by trypsinization after 24-h incubation. Cell pellets were solved in lysis buffer containing 7-M urea, 2-M thiourea and 4% CAHPS. After sonication, 0.05 mg of total protein was loaded onto immobilized pH 3–10 (linear) IPG strip (Amersham Bioscience, Arlington Heights, IL, USA) at 20 8C using a Ettan IPG phor (Amersham Bioscience). The IPG strip were rehydrated overnight in a solution of 7 M urea, 2 M thiourea, 4% CHAPS, 45 mM DTT, 0.5% IPG buffer, 400-mM Tris and a trace of bromophenol blue prior to use. After rehydration for 12 h, IEF was carried out using the following conditions: (1) 100 V, 50 VH; (2) 300 V, 125 VH; (3) 500 V, 250 VH; (5) 1000 V, 1000 VH; and (6) 6000 V, 40,000 VH. Following IEF, the gel strip was first equilibrated for 15 min in the equilibration buffer containing 130 mM DTT, 6 M

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urea, 2% SDS, 0.375 M Tris–HCl, pH 8.8, and 20% glycerol. Then the gel strip was equilibrated for another in the same equilibration buffer, except that DTT was replaced with 135 mM iodoacetamide (Sigma). The second dimensional SDS-PAGE was performed in 12% acrylamide gels using the Protean Xi system (Bio-Rad, Philadelphia, PA, USA). Proteins were visualized by silver stain.

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and TANK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-7 (Cell Signaling Technology, Beverly, MA, USA) and b-actin (Sigma) in 1% skim milk in PBST overnight at 4 8C. After washing in PBST, the membranes were incubated with secondary antibodies, goat anti-rabbit or goat anti-mouse IgG (Santa Cruz Biotechnology) conjugated to horseradish peroxidase for 1 h at room temperature. Bands were visualized using ECL kit (Amersham Bioscience).

2.5. Gel analysis 2.8. RT-PCR The stained 2-D gels were scanned with a Bio-Rad Scanner. The image analysis and 2 D gel proteome database management were done using the PDQuest software 6.2.1 (Bio-Rad). For identification of proteins by mass spectrometry, matching was done between analytical silver-stained gels and preparative gels in order to correlate the precise position if the spots to be excised. 2.6. MALDI-TOF-MS The spot of interest were rehydrated with 15 ml of trypsin (Roche, Mannheim, Germany) solution (10 mg/ml in 25 mM Ammonium bicarbonate buffer (pH 8.0)) at 37 8C for 12–14 h and extracted using 50% Acetonitrile/5% TFA (Sigma). After removal of Acetonitrile by centrifugation in a Speed-Vac, the peptides were concentrated by using C18Zip-Tip (Millipore Corp., Bedford, MA, USA) and eluted with 2 ml of 100% acetonitrile and directly spotted on the sample plate of a MALDI-TOF-MS. Finally, a-cyano-4-hydroxycinnamic acid (0.5 ml of 10 mg/ml) was applied to each spot, and the spots were air-dried at room temperature prior to acquiring mass spectra. Peptide mass profiles produced by MALDI-TOF were analyzed using MS-FIT and Mascot. Peptide masses were compared with the theoretical masses derived from the sequences contained in SWISS-PROT and NCBI data banks. 2.7. Western-Blot Proteins were separated on 12% SDS-PAGE gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in PBST (0.5% Tween-20 in PBS) and incubated with primary antibodies [i.e. caspase-3, -5, -8, CDK5, Fas (CD95)

The total RNA was isolated by using RNeasyw Midi kit (Qiagen, Hilden, Germany). Reverse trasncription was carried out at 50 8C for 30 min followed by 94 8C for 2 min and first round PCR for 30 cycles of 95 8C for 1 min, 56 8C for 1 min, 68 8C for 1 min and a final extension at 68 8C for 7 min. Primers used in this study were as follows. calpain 11 (5 0 -GGGATGCTGGCTCACATAAACAA-3 0 and 5 0 -GGGAAGGGAGCCGATGGCAG-3 0 ), SAP18 (5 0 -GGGATGGCGGTGGA GTCGCG-3 0 and 5 0 -GG GTTAATATGGTCTCAT-GCGCC-3 0 ), cdc2L5 (5 0 -GGG ATGCTGCCTGAAGATAAAGA-3 0 and 5 0 -GGGCTGCCGGAGAATTTTAATTT C-3 0 ), 0 Testin (5 -GGGAT-GGACCTGGAAAACAAAGT3 0 and 3 0 -GGGCTAGC TCTTATATTTCTTCAC-3 0 and glyceraldehyde-3-phosphate dehydro-genase (GAPDH) (5 0 -CCATGTTCGTCATGGGTGT0 GAACCA-3 and 5 0 -GCCAGTAGA GGCAGGGATGATGTTC-3 0 ). mRNA levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used to normalize the amount of sample RNA. 2.9. ProteinChip SELDI analyses of cellular extracts Protein extracts prepared from cultured cells were analyzed using a weak cation-exchange (WCX2) and strong anion exchange (SAX2) ProteinChip array (Ciphergen Biosystems, Freemont, CA, USA). The ProteinChip arrays were assembled into a deep-well type Bioprocessor assmbly (Ciphergen Biosystem). Prior to sample loading, WCX2 and SAX2 array were equilibrated with 150 ml of binding buffer (50 mM sodium acetate, pH 4.5 for WCX2 and 50-mM Tris– HCl, pH 8.5, for SAX2). The sample diluted in binding buffer was spotted onto each of following ProteinChip arrays. The arrays were then incubated

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with vigorous shaking for 30 min at room temperature. Sample was removed and the wells were individually washed with 350 ml binding buffer for 5 min. The arrays were washed twice with binding buffer for 5 min. Finally, the arrays were removed from the Bioprocessor and rinsed twice with 8 ml of distilled water in a 15-ml conical centrifuge tubes. After drying, 0.5 ml of saturated energy absorbing molecule solution [a-cyano-4-hydroxycinnamic acid in 50% acetonitrile (v/v), 0.5% trifluoroacetic acid (v/v)] was added two times and allowed to air-dry. Mass spectrometry analysis was performed by timeof-flight mass spectrometry in a PBS II mass reader (Ciphergen Biosystem). Spectra were collected using an average 80 nitrogen laser shots. Spectrum analysis

was performed using the ProteinChips software version 3.0 (Ciphergen Biosystems).

3. Results 3.1. Apoptosis by UA treatment in HeLa cervical carcinoma cells The presence of apoptotic cells was examined by DAPI staining. As shown in Fig. 1A, the apoptotic cells were characterized by cellular shrinkage, membrane blebbing and nuclear condensation. Nuclei were also brightly and uniformly stained by DAPI.

Fig. 1. (A) Nuclear morphology of DAPI stained cells was examined by fluorescence microscopy. (B) Cell-cycle progression of UA-treated HeLa cells treated with UA. Cell content of fixed and PI-stained cells was measured by flow cytometry and cell cycle was analyzed. M1 (%) represents portion of Sub G1 (hypodiploid DNA).

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The effect of UA on cell-cycle progression in HeLa cells studied after 24 h of drug exposure. In HeLa cells, the proportion of cell cycle was shown an outstanding difference between before and after treatment. UA treatment resulted in accumulation (Sub G1: 1.08%/10.63%) of HeLa cells in the Sub G1 phase (Fig. 1(B)). M1 (%) represents portion of Sub G1 (hypodiploid DNA). The FACS analysis demonstrated the cell-cycle arrest was occurred in apoptosis phase by treatment with UA. For quantitative analysis of mitotic arrest, the percentage of mitotic

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cells was calculated after counting at least 1!104 cells. 3.2. Proteome analysis to identify changes in protein expression stimulated by UA treatment in HeLa cervical carcinoma cells To analyze the underlying mechanisms that are unique to the UA treatment, we performed a proteome analysis to identify target-specific proteins important for enhanced apoptosis in cervical cancer cells.

Fig. 2. (A) 2DE maps of DMSO-treated HeLa (control) and UA-treated HeLa cervical carcinoma cells. (B) 2DE and differential protein expression analysis under apoptotic condition. Proteins spots marked on the maps were considered differentially expressed and identified by MS.

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levels were either increased or decreased in UA-treated HeLa cells. Proteins spots marked on the maps were considered differentially expressed and identified by MS (Fig. 2(B)). Among them, 18 proteins were upregulated and seven proteins down-regulated by UA as determined by spot volume (P!0.05) (Tables 1 and 2). Twenty-five of 45 proteins were successfully identified by MALDI-TOF-MS and database searches with high confidence based on high scores and sequence coverage. The identified up-regulated proteins were as follows; sin3 associated-polypeptide p18 (SAP18), transcriptional adapter 2-like, eukaryotic initiation factor 4A-like NUK-34, eukaryotic translation initiation factor 3 subunit 2, phosphatidylinositol-4phosphate 5-kinase type II a, tRNA pseudouridine synthase A, D-beta-hydroxybutyrate dehydrogenase, TRAF family associated NF-kb activator (TANK),

Using high-resolution two-dimensional gel electrophoresis (2DE), we determined the protein expression profile of HeLa cells after UA treatment. An expression profile map from UA-treated HeLa cells was used to compare with a master expression profile map of DMSO-treated control cells. This master pattern of protein expression was used to identify difference after UA treatment. Three pairs of gels from different batches of DMSO- and UA-treated cells were analyzed for quantitative spot comparisons with the image analysis software. An expression profile map from UA-treated cells was used to compare with a master expression profile map of DMSO-treated control cells (Fig. 2(A)). More than 45 proteins showed significant changes O2.5-fold in UA-treated cells compared to control cells. Twentyfive proteins were affected by treatment of UA, as their Table 1 Up-regulated proteins by UA treatment in HeLa cervical carcinoma cells Accession no.

Protein

Function

Mass (Da)/pI

O00422

Sin3 associated-polypeptide p18

17561/9.4

Q75478

Transcriptional adapter 2-like

P38919

Eukaryotic initiation factor 4A-like NUK-34

Q13347

Eukaryotic tralslantion initiation factor 3 subunit 2

P48426 Q9Y606

Phosphatidylinositol-4-phosphate 5-kinase type II alpha tRNA pseudouridine synthase A

Q02338

D-beta-hydroxybutyrate

Q92844

TRAF family associated NF-kb activator (TANK)

P25445 Q9UMQ6 NP11678 P51878 P55210

TNF-receptor superfamily member 6 precursor (Fas) Calpain 11) Caspase-3 Caspase-5 precursor Caspase-7 precursor

NP203520

Caspase-8

NP004926 Q9UG18 Q14004

CDK5 (cyclin-dependent kinase 5) Testin (TESS) Cell division cycle 2-like protein kinase 5 (CDC2L5) B-cell receptor-associated protein 31

Enhances the ability of Sin3-HDAC1-mediated transcriptional repression. Associated with the P/CAF protein in the PCAF complex. ATP-dependent ssDNA-binding protein with a sequence-undependent unwinding activity (Helicase) Binds to the 40S ribosome and promotes the binding of methonyl Catalyzation of the phosphorylation of phosphatidyl-inositol-4-phosphate Converts specific uridines to PSI in a number of tRNA substrates (R)-3-hydroxybutanoateCNAD(C)Z acetoacetateCNADH Overexpression inhibits TRAF2-mediated NF-kb activation signaled by CD40 Receptor for TNFSF6/FASL. The adaptor molecule FADD recruits caspase-8 to the activated receptor. Calcium-regulated non-lysosomal thiol-protease Cleavage and activation caspases 6, 7 and 9 Mediator of apoptosis Involved in the activation cascade of caspases responsible for apoptosis excution. Programmed cell death induced by Fas and various apoptotic stimuli Probably involved in the control of the cell cycle May act as a tumor suppressor May be a controller of the mitotic cell cycle involved May be involved in CASP8-mediated apoptosis

P51572

dehydrogenase

51496/6.5 46871/6.3

23354/4.9 46224/6.5 44378/7.6 38303/8.9 47624/5.6 37733/8.3 80583/5.6 34532/10.3 47815/9.2 34277/5.7 20836/11 34413/7.9 47997/8.0 48212.8.3 27992/8.4

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Table 2 Down-regulated proteins by UA treatment in HeLa cervical carcinoma cells Accession no.

Protein

Function

Mass (Da)/pI

P06727

Apolipoprotein A-IV precursor (Apo-AIV)

45371/5.3

P54750

Calcium/calmodulin-dependent 3 0 ,5 0 -cyclic nucleotide phosphodiesterase 1A (hcam-1)

Q9BW91

ADP-ribose pyrophosphatase (ADPR-PPase)

Q9H939

Proline-serine-threonine phosphatase-interacing protein 2 Synapsomal-associated protein 23 (SNAP-23)

May have a role in chylomicrons and VLDL secretion and catabolism. Requored for efficient activation of lipoprotein lipase by APOC-11. Has a higher affinity for cGMP than for cAMP. Nucleoside 3 0 ,5 0 -cyclic phosphateCH(2)OZ nucleoside 5 0 -phosphate ADP-riboseCH(2)OZAMPCD-ribose 5-phosphate. Binds to F-actin. May be involved in regulation of the actin cytoskeleton Essential component of the high affinity receptor for the general membrane fusion machninery and an important regulator of transport vesicle docking and fusion. Binds to the 40S ribosome and promotes the binding of methonyl Endopeptidase that degrades various components of the extracellular matrix, such as fibrin. May be involved in the activation of membrane-bound precur sors of growth factors or inflammatory mediators, such as tumor necrosis factor-alpha

O00161

Q13347 Q9ULZ9

Eukaryotic translation initiation factor 3 subunit 2 (TGF-beta receptor interacting protein 1) Matrix metalloproteinase-17 precursor (MMP17)

TNF-receptor superfamily member 6 precursor (Fas), calpain 11, caspase-3, caspase-5 precursor, caspase-7 precursor, caspase-8, CDK5 (cyclin-dependent kinase 5), Testin (TESS), Cell division cycle 2-like protein kinase 5 (CDC2L5), B-cell receptor-associated protein 31. And the down-regulated proteins were as follows; apolipoprotein A-IV precursor (Apo-AIV), calcium/calmodulin-dependent 3 0 , 5 0 -cyclic nucleotide phosphodiesterase 1A (hcam-1), ADP-ribose pyrophosphatase (ADPR-PPase), proline-serinethreonine phosphatase-interacing protein 2, synapsomal-associated protein 23 (SNAP-23), eukaryotic translation initiation factor 3 subunit 2 (TGF-beta receptor interacting protein 1), matrix metalloproteinase-17 precursor (MMP17). The identified proteins were mostly involved in apoptosis; caspase-3, -5, -7, fas (CD95), calpain and cyclin dependent kinase 5 (CDK5). Western blot and RT-PCR were performed for proteins to confirm increasing expression data derived from proteomics: caspase family (caspase-3, -5 and -7) (Fig. 3A), CDK 5, fas and TANK (Fig. 3B), calpain 11, SAP18, cdc 2L5 and testin (Fig. 3(C)). Expression data obtained with 2DE proteomics were strongly correlated with the western blot and RT-PCR methods, respectively.

61252/5.7

39125/8.3 38701/8.7 23354/4.9

36502/5.4 67007/6.1

3.3. ProteinChip analysis The SELDI mass spectrum (!20 kDa) was revealed different protein profiles of the two cell samples (Fig. 4). Most of protein peaks in cell extracts of control and UA-treated HeLa cells are identical. The representative protein profiles obtained on WCX2 chip are shown in Fig. 5 as mass spectrum. Analysis of UA treated HeLa cell relative to control revealed that two peaks were significantly differentially expressed proteins was 8562.9 and 10069.1 Da (P!0.05) (Fig. 5). To search for a hint the protein identities of two proteins, 8562.9 and 10069.1 Da, we used the TagIdent tool from the ExPASy molecular server. By entering the mass of an unknown protein, this tool will search in the SWISS-PROT and TrEMBLE protein databases for proteins that will match with requested mass. We found calcium/ calmodulin-dependent protein kinase II inhibitor a matches the protein peak with a mass of 8562.9 Da. The function of this protein is to regulate calciumdependent signaling. cytochrome c oxidase, polypeptide VIb (AED) matches the protein peak with a mass of 10069.1 Da. This protein is one of the nuclearcoded polypeptide chains of cytochrome c oxidase,

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Fig. 3. Differentially expressed proteins from proteomic analysis were identified by western blots and RT-PCR. (A) Comparisons of the proteins (caspase-3, -5, -7, and -8) obtained from DMSO-treated HeLa (control) and UA treated HeLa cells. (B) Comparison of the proteins (CDK5, fas and TANK) obtained from DMSO-treated HeLa (control) and UA-treated HeLa cells. b-actin was used to normalize protein loading. (C) It revealed increasing pattern of mRNA of Calpain 11, SAP18, CDC2L5 and testing. GAPDH mRNA level was used as a control.*:cleavage form, **:full length form.

the terminal oxidase in mitochondrial electron transport. A representative spectrum of sample on SAX2 arrays is shown in Fig. 6. Expression profiles between the samples revealed several protein patterns differences and a 6014.4, 8545.91, 10099.6, 11604.1, 14552.1 and 11648.2 Da proteins were found as a highly expressed peak. The protein peak 3129.9, 5132.5, 7208.3 and 14552.1 Da dramatically decreased in UA-treated HeLa cells (Fig. 6). By using SWISS-PROT and TrEMBLE protein database, we found pyrin-only protein 1 matches the protein peak with a mass of 10099.6 Da. Pyrin-only protein regulates inflammatory signaling. The remaining increased peaks (6014.4, 8545.91, 11604.1, 14552.1 and 11648.2 Da) were unsuccessfully identified. Signal transducer CD24 precursor matches the protein peak with a mass of 3129.9 Da and modulates B cell activation response. Also, the protein promotes antigen-dependent proliferation of B cells and prevents their terminal differentiation into antibodyforming cells. NIK-associated protein, a novel silencer of TNF and IL-1-induced NF-kB activation, matches the protein peak with a mass of 7208.3 Da. Cystatin-like metastasis-associated protein matches the protein peak with a mass of 14552.1 Da and

the function may play a role in immune regulation. The remaining decreased peaks (3129.9 and 5132.5 Da) were unsuccessfully identified.

4. Discussion UA has been reported that UA induces: (i) cellcycle arrest concomitantly with the apparition of the apoptotic sub-group G1 peak, and (ii) cell death through apoptosis, which is mediated by caspase-3 in HaCat cell [25]. Very little is known about the signaling pathways mediating UA-induced cell death. We performed proteome analysis including 2DE/MALDI-TOF-MS and SELDI-TOF-MS to find other proteins participated in the process of UAinduced apoptosis in HeLa cervical carcinoma cells. UA-induced apoptosis was characterized by cellular shrinkage, membrane blebbing and nuclear condensation. UA treatment resulted in accumulation of HeLa cells in the Sub G1 phase on cell-cycle progression. Apoptosis could be induced via the activation of the fas (CD95)-dependent manner by UA treatment. The binding of fas ligand to its Fas/APO-1/CD95 cell-surface death receptor triggers the activation of caspase-8. Therefore it can directly cleave procaspase-3 in its

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Fig. 4. Representative protein spectra (upper panel) and gel view (lower panel) of SELDI analysis (!20 kDa) of cellular protein bound to WCX2, SAX2 ProteinChip array.

activated form. Our results showed UA could induce caspase-8 and -3 processing and apoptosis in a CD95dependent manner HeLa cells. CDK5, which is a serine/threonine kinase, has high sequence similarity to the cell cycle regulating CDK family members, but it is neither activated by cyclins nor involved in cell-cycle regulation [26,27]. The common general function of the CDK family members is to ensure the normal progression through the cell cycle, and they are tightly regulated by the sequential expression of cyclins. The unscheduled activation of cell cycle-related CDKs such as CDK1 and CDK2 might have an impact late in apoptosis since they are activated by caspases [28]. In the present studies, CDK5 showed the increasing expression pattern in UA-treated HeLa cells. Calpain is a family of calcium-dependent cysteine proteases of which two isozymes, m- and m-calpain, are expressed ubiquitously. These enzymes are

heterodimeric and consist of an 80-kDa catalytic subunit and a 30-kDa subunit whose function is unclear. In contrast to caspases, calpain does not appear to have strict sequence requirements for substrate cleavage [29]. Although caspases play a major part in the demise of cells that have been triggered to undergo apoptosis, there is evidence that other proteases including calpain may also be involved in this process. We confirmed increasing calpain 11 mRNA expression level by using RT-PCR. TANK (TRAF family associated NF-kb activator) inhibits LMP1-mediated NF-kb activation. Overexpression of TANK enhances apoptosis by activating cell death signals and inhibiting survival signals [30]. SAP18 (Sin3 associated-polypeptide p18), component of the Sin3-repressing complex, enhances the ability of Sin3-HDAC1-mediated transcriptional repression. When tethered to the promoter, it can direct the formation of a repressive complex to core

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Fig. 5. SELDI mass spectrum for differentially expressed molecules on a WCX2 ProteinChip array. Analysis of UA-treated HeLa cell relative to control revealed that two peaks were significantly differentially expressed proteins was 8562.9 and 10069.1 Da.

histone proteins [31]. Therefore, Overexpression of SAP18 mRNA means repression of cellular transcription by UA. CDC 2L5 (Cell division cycle 2-like protein kinase 5) may be a controller of the mitotic cell

cycle [32]. In RT-PCR results of overexpression by UA treatment, this also may involve in cell-cycle regulation like CDK family. Testin, which may act as a tumor suppressor, showed increasing expression pattern of Testin

Fig. 6. SELDI mass spectrum for differentially expressed molecules on a SAX2 ProteinChip array, whereas the peak 6014.4, 8545.91, 10099.6, 11604.1, 14552.1 and 11648.2 Da proteins were found as a highly expressed peak, the peak 3129.9, 5132.5, 7208.3 and 14552.1 Da were decreased in UA-treated HeLa cell.

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mRNA in our results. Although Testin mRNA was expressed in all normal human tissues, lack of expression of this gene was found in several cancerderived cell lines [33]. These results summarized as follows. First, UA seems to induce caspase-8, -3 processing and apoptosis in a Fas-dependent fashion: UA/ caspase-8/caspase-3/apoptosis. We suggest that, in HeLa cells, UA may require fas receptor function to induce caspase-8, -3 processing and apoptosis. Second, UA induces the CDK5 overexpression which mediates spontaneous apoptosis. Third, calpain may play a central role in the execution of apoptosis either upstream or downstream of caspase in UA-treated HeLa cells. Interestingly, caspases and calpains shared several substrates (e.g. endogenous calpain inhibitor calpastatin, focal adhesion kinase, calmodulin-dependent kinases). This indicates that these enzymes may execute the cell in a partially indistinguishable manner. Thus, our results suggest that activity of caspase, calpain and CDK5 may play an important role during the degradation phase of the apoptotic program in UA-induced apoptosis model. In this study, we attempted to use SELDI-TOF MS to characterize and identify of several specific proteins, which are increased or decreased by UA in cervical carcinoma cell lines. Processing on a WCX2 ProteinChip, calcium/calmodulin-dependent protein kinase II inhibitor a and cytochrome c oxidase, polypeptide VIb (AED) proteins were increased by UA treatment. The function of calcium/calmodulindependent protein kinase II inhibitor a is to regulate calcium-dependent signaling and cytochrome c oxidase, polypeptide VIb (AED) is associated with mitochondrial electron transport. In SAX2 ProteinChip analysis, pyrin-only protein 1 was increased, whereas signal transducer CD24 precursor, NIK-associated protein and cystatin-like metastasisassociated protein were decreased by UA treatment. The pyrin-only protein1 regulates inflammatory signaling, signal transducer CD24 precursor promotes ag-dependent proliferation of B cells and prevents their terminal differentiation into antibody-forming cells, NIK-associated protein is a novel silencer of TNF and IL-1-induced NF-kB activation and cystatinlike metastasis-associated protein plays a role in immune regulation. We failed to find matched proteins between 2DE/MALDI-TOF-MS and SELDI-TOF-MS

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results because of the proteins or peptides from SELDI-TOF-MS are low molecular mass because of the specific ionization and chip surface conditions used (WCX2 and SAX2 ProteinChip). In conclusion, we have demonstrated by using a 2DE/MALDI-TOF-MS and ProteinChip-based technology that several specific proteins are increased or decreased by UA treatment The emergence of new technologies for the identification of unknown proteins from mass spectrometry profiles is expected to accelerate the identification and characterization of these proteins that will reveal the molecular basis of UA-induced apoptosis.

Acknowledgements This work was supported by the Korea Science and Engineering Fund through the Cancer Metastasis Research Center (CMRC) at Yonsei University (R11-2000-082-02007-02003). S.J.U. was supported by BK21 project from the Ministry of Education and Human Resources Development.

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