Protein profiling and identification of modulators regulated by human papillomavirus 16 E7 oncogene in HaCaT keratinocytes by proteomics

Gynecologic Oncology 99 (2005) 142 – 152 www.elsevier.com/locate/ygyno

Protein profiling and identification of modulators regulated by human papillomavirus 16 E7 oncogene in HaCaT keratinocytes by proteomics Kyung-Ae Lee a,1, Jeong-Woo Kang a,1, Jung-Hyun Shima, Chang Won Kho b, Sung Goo Park b, Hee Gu Leea, Sang-Gi Paik c, Jong-Seok Lim a,2, Do-Young Yoon a,* a

Laboratory of Cellular Biology, Korea Research Institute of Bioscience and Biotechnology, Yuseong, P.O. Box 115, Daejeon 305-600, South Korea Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yuseong, P.O. Box 115, Daejeon 305-600, South Korea c Laboratory of Cellular Biology, Chungnam National University, 220 Gung-dong, Yuseong, Daejeon 305-764, South Korea

b

Received 21 December 2004 Available online 20 July 2005

Abstract Objectives. Viral oncogenes E6 and E7 are selectively retained and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperated with each other in immortalization and transformation of primary keratinocytes. This study was performed to identify proteins to be bound or modulated by high risk HPV E7 oncogene by using a proteomics. Methods. HaCaT normal keratinocyte was prepared to establish a stable cell line expressing E7. The E7-affinity column was also prepared to obtain E7-interacting proteins. In order to search the target molecules modulated by E7 expression, we used 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight (MALDI/TOF) mass spectrometry. Pull down assay was also performed in order to confirm the E7-interacting proteins. Results. We identified 28 spots that are modulated by E7 in HaCaT/E7 using 2-dimensional electrophoresis (2-DE) and MALDI/TOF mass spectrometry. Proteomics analyses showed that actin and leukocyte elastase inhibitor were down-regulated, whereas stress-induced phosphoprotein 1, CD2 binding protein 1, catalase, T-complex protein 1, Ku70-binding protein, heat shock 60 kDa protein 1, G1/Sspecific cyclin E1 and peroxiredoxin 2 were up-regulated. Western blot revealed that heat shock 60 kDa protein, catalase and peroxiredoxin 2 were also up-regulated. Pull down assay also showed that leukocyte elastase inhibitor (LEI) and Ku70-binding protein were bound to the E7 oncoprotein. By using E7-affinity column and 2-DE/MALDI-TOF, 22 spots were found to interact with E7 recombinant protein. MG11-like proteins, livin inhibitor-of-apoptosis, protein serine kinase c17, CD2 binding protein 1, cyclin E1, TATA box binding protein-associated factor and uridine – cytidine kinase 2 were up-regulated by E7 oncogene and also bound to E7 oncoprotein. Conclusions. It is presumed that E7 can influence cell status by modulating the factors related to cell signaling, apoptosis and cell cycle regulation. D 2005 Elsevier Inc. All rights reserved. Keywords: Proteomics; Two-dimensional gel electrophoresis; Affinity column; Human papillomavirus; E7

Introduction

* Corresponding author. Fax: +82 42 860 4593. E-mail address: [email protected] (D.-Y. Yoon). 1 Equally contributed to this manuscript. 2 Present address: Department of Biological Science, Sookmyung Women’s University, Hyochangwongil 52, Yongsan-ku, Seoul 140-742, South Korea. 0090-8258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2005.05.039

Cervical cancer is one of the leading causes of female death from cancer worldwide. Human papillomaviruses (HPVs) have been recognized as a primary cause of cervical cancer. Specific types of HPV (16, 18 and several others) have been identified as causative agents of at least 90% of cervical cancers and are linked to more than 50% of other anogenital cancers [1].

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

HPVs have circular double-stranded DNA genomes that are approximately 8 kb in size and encode eight genes of which E5, E6 and E7 have transforming properties [2– 4]. HPV 16 E7 protein can form a specific complex with phosphorylated pRb [5]. The release of E2F influences the expression of the genes involved in mitosis and cell cycle control [6]. Therefore, the formation of a complex between the products of oncogenes and the tumor suppressor genes is believed to be important in the cellular transformation that leads to the disruption of the normal physiological functions of the specific tumor suppressor gene products [7]. E7 oncoprotein also functions in a pRb-independent manner. E7 oncoprotein exhibits multiple functions through protein interactions with pRb as well as with other pocket proteins, cyclin-dependent kinase-2 and transcription regulating proteins, including the TATA box binding protein and AP-1 transcription factor [8,9]. From the obtained extensive knowledge of various molecular mechanisms by HPV oncogenes, we expected to find proteins related to immune escape mechanisms and apoptosis. Proteomics applied with the combination of 2-DE and MALDI/TOF mass spectrometry has recently been used as a new technology for identifying proteins in living organisms and for determining their pattern of expression [10,11]. These technologies can potentially provide answer to biological questions regarding the mechanisms involved in the pathogenesis of cancer. Although protein databases based on proteomics are available for several cancer researches, no database exists for cervical cancer induced by HPV E7 oncogene except several recent reports. In addition to the proteomic approach, we used E7-affinity column in order to find the novel mechanism of E7-associated cellular immune responses and to analyze the interaction between E7 and E7-binding proteins. We have purified recombinant his-tagged E7 oncoprotein and prepared E7-affinity column to obtain E7interacting proteins. The E7-interacting proteins were resolved on 2D-gel and analyzed by MALDI/TOF. By using 2-DE, we examined whether high-risk HPV 16 E7 oncogene can affect or bind to the factors on the track of immune system to elucidate the possible immune escape mechanism of high risk HPV-infected cervical cancer. We identified chaperones, cell cycle regulatory factors, immune modulating factors, cell signaling factors and novel factors which are shown to be modulated by E7 oncogene.

143

Biotechnology, Santa Cruz, CA, USA), anti-hsp60 and anti-HOP (Stressgen Bioreagents, Glanford Avenue), antih actin and horseradish-peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, USA) and anti-flag (Affinity BioReagents, Suite Golden, CO). Polyclonal anti-human Ku70BP antibody was made and used (KRIBB, Daejeon, Korea). Cell culture HPV-positive cervical cancer cell lines, such as CaSki, HeLa, human normal HaCaT keratinocytes and HEK293 embryonic kidney cells, were maintained in DMEM supplemented with 100 U/ml penicillin, 100 Ag/ml streptomycin, 25 ng/ml amphotericin B (Gibco BRL, Grand Island, NY, USA) and 10% fetal bovine serum (FBS) (Hyclone, South Logan, UT) at 37-C in a humidified incubator with 5% CO2. CaSki cell lines have been known to contain 60– 600 copies of HPV 16 genome [12]. HaCaT was used to establish mock control- and E7expressing stable cell lines as previously reported [13]. HaCaT cells transfected with E7 oncogene were selected in a selection DMEM medium containing G-418 as previously described [16]. HaCaT/E7 cells were cultured in a selection medium containing G-418 every month in order to maintain multiple clones. HEK293 was used to establish leukocytes elastase inhibitor (LEI)-expressing stable cell line. Plasmid construction and expression of protein E7 was inserted into pET28a (Invitrogen, Carlsbad, CA) and expressed in Escherichia coli as an N-terminal histagged recombinant protein, as previously described [14]. GST-tagged E7 was inserted into pGEX-4T-1 (Amersham Pharmacia Biotech, Little Chalfont, UK) and expressed in DH5a [15]. Two micrograms of total RNA from HepG2 cells was reverse-transcribed by RT-PCR, and then the resulting cDNAs were PCR-amplified with human leukocytes elastase inhibitor (LEI) gene- or Ku70BP genespecific primers sets. LEI cDNA was flag-tagged at the C-terminal by cloning it into pCMVtag4A (Stratagene, La Jolla, CA). Ku70BP cDNA was cloned into pCR2.1-TA cloning vector (Invitrogen) and then transferred to pET28a vector (Invitrogen). pET28a/Ku70BP was expressed in E. coli BL21 (DE3) and purified with Ni-NTA as previously described [15]. Establishment of stable transfectants

Materials and methods Antibodies The following were purchased: anti-TCP-1, anti-E7, horseradish-peroxidase-conjugated anti-rat IgG and horseradish-peroxidase-conjugated anti-goat IgG (Santa Cruz

The pOP13 vector expressing E7 was transfected into HaCaT, and stable transfectant was selected by growing it in the selective DMEM containing 5 Ag/ml of G-418, as previously described [16]. Five micrograms of pCMVtag4ALEI was transfected into HEK293 cells using GENESHUTTLE (Qbiogene, Morgan Irvine, CA).

144

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

Establishment of E7-affinity column Histidine fusion proteins were expressed in E. coli BL21 (DE3) using pET28a/E7 and bound to affinity column as previously described [17]. GST fusion proteins were expressed in DH5a using pGEX-4T-1/E7 and bound to GST agarose as previously described (Amersham Pharmacia Biotech) [15]. Sample preparation for two-dimensional gel electrophoresis Cells from 100 mm dishes were harvested, washed with PBS and then lysed in a lysis buffer (25 mM Tris – phosphate, 2 mM DTT, 2 mM 1,2-diaminocyclohexaneN,N,NV,NV-tetraacetic acid, 10% glycerol, 1% Triton X-100, pH 7.8) (Promega, USA) containing aprotinin (10 Ag/ml) and 0.5 mM phenylmethylsulfonyl fluoride. The cell lysates were obtained by centrifugation at 12,000  g for 30 min after incubation on ice for 30 min and dialyzed against 20 mM Tris – HCl, pH 8.0 for 20 h. Sample preparation by combining E7-affinity column with HaCaT/E7 and mock control cells lysates in a binding buffer (50 mM NaH2PO4, 300 mM NaCl) containing 10 mM imidazole was performed. The cell lysates (2– 4 mg) were obtained by centrifugation at 12,000  g for 30 min after lysis on ice for 30 min and applied to the E7-affinity column (100 Al). The mixtures were rotated in microtubes at 4-C for 1 h. The cellular proteins bound to E7-affinity column were washed three times with a binding buffer containing 20 mM imidazole and then eluted with 200 mM imidazole for 30 min. Eluted proteins were precipitated with 10% TCA and dialyzed against 20 mM Tris – HCl, pH 8.0 for 20 h. The protein concentration was determined by Bradford method. Two-dimensional gel electrophoresis For the first dimension, pH 3 – 10 immobilized pH gradient (IPG) gel strips (13 cm; Amersham Pharmacia Biotech) were rehydrated overnight in a rehydration solution containing 250 Ag of protein sample in an IPGphor strip holder covered with a cover fluid. Isoelectric focusing was conducted at 20-C using an IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech). A three-phase program was used for the analytical gel. The first phase was set at 1000 V for 1 h, the second phase was set at 2000 V for 2 h, and the third phase was a linear gradient spanning from 2000 V to 8000 V for 14 h. Prior to the 2-DE, the IPG gel strips were equilibrated for 15 min in an SDS equilibration solution (50 mM Tris –Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, 60 mM dithiothreitol). The second-dimensional separation was carried out on 12% SDS-PAGE gel (16  20 cm) without stacking gel at 4-C. The IPG strips were embedded on top of the gel with 1% agarose. Electrophoresis was carried out at 60 mA/gel for 6 h until the bromophenol blue reached the bottom of the gel. The gel was

fixed and then silver-stained according to the manufacturer’s procedure (Amersham Pharmacia Biotech). The gel was stained with silver for 2 h and then destained as described below and previously [18]. Destaining Silvers were removed from the gel using chemical reducers as described elsewhere [19]. In brief, working solution was prepared by mixing at a ratio of 1:1 of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. Thirty to fifty microliters of working solution was added to cover the gel for 20 min and then discarded. Subsequently, the gel was cut into small pieces, washed with water and dehydrated repeatedly with changing acetonitrile until the gel pieces turned opaque white. The gel pieces were then dried in a vacuum centrifuge for 30 min. Trypsin digestion of proteins in the gels was performed as described elsewhere [20]. MALDI-TOF mass spectrometric analysis and database search Each mass spectrometric analyses were performed using a PerSeptive Biosystems MALDI-TOF Voyager DE-RP mass spectrometer (Framingham, MA) operated on the delayed extraction and reflector mode. Peptide mixtures were analyzed by using the saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile/ 0.1% trifluoroacetic acid [21]. Database search was performed using PEPTIDENT of the program ExPASY (http://cn.expasy.org/tools/peptident.html). Western blot analysis Cellular proteins and E7-binding proteins used for 2-DE and MALDI-TOF were electrophoresed in 12% SDSPAGE, transferred to PVDF membrane (Millipore, Bedford, MA) and incubated with antibodies overnight at 4-C followed by 1 h incubation with respective secondary antibodies conjugated with horseradish peroxidase and developed by ECL (Amersham Pharmacia Biotech). In vitro pull down assay Binding assays were performed by combining GST-E7 immobilized on glutathione –Sepharose with cell lysates from LEI transfectants lysed in a lysis buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 0.1% NP-40, 0.1% Triton X-100, 1 mM EDTA) or purified his-tagged Ku70BP. The reaction mixtures were washed three times with PBS containing 0.5% Triton X-100, separated by SDS-PAGE and transferred onto the PVDF membrane. Ku70BP and E7 protein were detected by mouse polyclonal anti-Ku70BP and mouse monoclonal anti-HPV 16 E7 antibody, respectively. LEI-flag was detected by using an anti-flag antibody. After the membrane was used for LEI-flag signal, it was stripped to detect E7.

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

Results Two-dimensional electrophoresis patterns of proteins from HaCaT after transfection with E7 and identification of E7-modulated proteins by mass spectrometry Protein profiling was conducted to identify proteins modulated by E7 using 2-D gel electrophoresis and silver staining. In order to elucidate the specific effects of E7

145

oncogene on cells, we compared the differential patterns of 2D between mock vector and E7-transfected cells (Fig. 1). We did not compare the differential 2-D patterns between the non-transfected and the mock control vector-transfected HaCaT. Although there are some spots shown to be downor up-regulated by E7, we selected those spots, separately (Fig. 1). Twenty-eight protein spots were identified from the selected parts of the 2-D gels. Among the 28 spots, 2 spots were down-regulated, whereas 26 were up-regulated by E7

Fig. 1. Two-dimensional electrophoresis patterns of proteins from HaCaT after transfection with E7 or mock vector. The proteins were separated by 2dimensional electrophoresis and stained with silver nitrate. Identified protein spots are indicated by numbers. Down-regulated proteins by E7 expression are indicated in the left panel (mock control), and up-regulated proteins are indicated in the right panel (E7-transfected cells). The numbers indicate the position of the up- and down-regulated protein spots in 2-D pattern by E7. The x axis represents pH 3 – 10 immobilized pH gradient for the first dimension, and y axis represents the molecular weight of standard markers.

146

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

Table 1 Identification of E7-suppressed or -induced protein spots by mass spectrometry Spot A 1

Score

# peptide matches

AC

Description

pI

Mw

Arbitrary intensity

CHAIN 1: ACTIN, CYTOPLASMIC 1 CHAIN 1: ACTIN, CYTOPLASMIC 2 Leukocyte elastase inhibitor (LEI) (monocyte/neutrophil elastase inhibitor) (M/NEI) Unknown (protein for IMAGE:4301014) (fragment) Hypothetical 27.1 kDa protein (fragment)

5.29 5.31 5.9

41,605.54 41,661.65 42,741.81

+++

Undefined Undefined

Undefined Undefined

6.4

62,639.26

6.32

63,254.72

5.31 Undefined 6.95 5.42 6.66

65,568.63 Undefined 59,624.98 49,880.21 64,256.13

5.93 7.55

64,066.56 59,366.62

+ +

Undefined

Undefined

+

7.05 Undefined 6.29

52,971.87 Undefined 56,650.5

++

6.29 5.5 4.75 6.13

56,664.53 59,812.09 49,758.9 43,786.86

++ + ++

6.53

46,659.3

++

5.29

53,110.4

6.29

37,025.4

6.57

46,116.31

+

5.7 5.23 Undefined Undefined 4.63

47,077.19 36,399.61 Undefined Undefined 29,173.9

++ ++ ++ ++

Undefined 4.56

Undefined 26,599.07

++

Undefined 6.12 5.84 4.8

Undefined 24,393.52 24,772.77 28,171.4

6.66 Undefined

23,566.79 Undefined

0.33 0.33 0.41

6 6 9

P02570 P02571 P30740

0.18 0.18

4 4

Q96I31 Q9UF45

0.36

8

P31948

0.23

5

0.19 0.19 0.15 0.22 0.17

5 5 5 5 4

5 6

0.11 0.29

4 10

P11171 (isoform) O75419 Q9HC48 P04040 P14136 Q05682 (isoform) P36406 Q99832

7

0.21

6

Q9P2I0

8

0.21 0.14 0.16

6 4 7

O60687 Q9Y6H3 Q9BQ01

9 10 11

0.16 0.39 0.43 0.23

7 9 10 5

O43175 Q96FZ6 P07437 Q9UQ80

12

0.44

12

Q9UKW8

0.15

4

Q9ULV5

0.15

4

P51665

13

0.27

8

P17174

14 15 16 17

0.13 0.19 0.11 0.22 0.14

4 4 5 5 6

P24864 Q9NR44 Q9Y6H3 Q9NTD1 P42655

18

0.12 0.1

5 4

Q29909 P56537

20 21

0.08 0.08 0.24 0.08

4 4 4 4

Q9Y6H3 P24410 Q8TD75 P35214

22

0.08 0.21

4 4

P11233 Q9UFU2

2

B 1

2 3 4

19

Stress-induced-phosphoprotein 1 (STI1) (hsp70/hsp90-organizing protein) (transformation-sensitive protein IEF SSP 3521) SPLICE NON-ERYTHROID isoform B of protein 4.1 (Band 4.1) (P4.1) (EPB4.1) (4.1R) CDC45-related protein (PORC-PI-1) CTCL tumor antigen se2 – 5 (fragment) Catalase (EC 1.11.1.6) Glial fibrillary acidic protein, astrocyte (GFAP) SPLICE isoform 3 of caldesmon (CDM) GTP-binding protein ARD-1 (tripartite motif protein 23) T-complex protein 1, eta subunit (TCP-1-eta) (CCT-eta) (HIV-1 Nef interacting protein) Cleavage and polyadenylation specificity factor, 100 kDa subunit (CPSF 100 kDa subunit) (fragment) Sushi-repeat protein Ku70-binding protein (fragment) Phosphoglycerate dehydrogenase (unknown) (protein for MGC:18226) D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) (PGDH) Heat shock 60 kDa protein 1 (chaperonin) Tubulin beta-1 chain Proliferation-associated protein 2G4 (cell cycle protein p38-2G4 homolog) (hG4-1) NADP+-dependent isocitrate dehydrogenase (unknown) (protein for MGC:21176) Heat shock factor protein 4 (HSF 4) (heat shock transcription factor 4) (HSTF 4) (hHSF4) 26S proteasome non-ATPase regulatory subunit 7 (26S proteasome regulatory subunit S12) (proteasome subunit p40) Aspartate aminotransferase, cytoplasmic (EC 2.6.1.1) (transaminase A) (glutamate oxaloacetate transaminase-1) G1/S-specific cyclin E1 Butyrophilin, subfamily 3, member A2 Ku70-binding protein (fragment) Hypothetical 34.7 kDa protein (fragment) 14-3-3 protein epsilon (mitochondrial import stimulation factor L subunit) (protein kinase C inhibitor protein-1) (KCIP-1) (14-3-3E) HLA-A24 (fragment) Eukaryotic translation initiation factor 6 (eIF-6) (B4 integrin interactor) (CAB) (p27 (BBP)) (B(2)GCN homolog) Ku70-binding protein (fragment) Ras-related protein Rab-11A (RAB-11) (24KG) (YL8) Delta 4-SF1b truncated prolactin receptor 14-3-3 protein gamma (protein kinase C inhibitor protein-1) (KCIP-1) Ras-related protein RAL-A Hypothetical 23.3 kDa protein (fragment)

++

+

+ ++ +

+ + +

++

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

147

Table 1 (continued) Spot

Score

23

0.2

24 26

0.15 0.08

# peptide matches

AC

Description

pI

Mw

Arbitrary intensity

5

P32119

5.66

21,891.92

+

7 4

Q9BUH5 P25912 (isoform)

Peroxiredoxin 2 (thioredoxin peroxidase 1) (thioredoxin-dependent peroxide reductase 1) (thiol-specific antioxidant protein) (TSA) (PRP) (natural killer cell enhancing factor B) (NKEF-B) Transgelin 2 (SM22-alpha homolog) SPLICE isoform 3 of max protein

8.41 5.64

22,406.42 12,099.12

++ +++

Down- or up-regulated proteins by E7 are described. The peptide profiles of the protein-spots treated with trypsin were analyzed by MALDI-TOF-MS and by using the Expasy Peptident search program.

(Fig. 1). The protein spots were excised from the gels, digested with trypsin and then analyzed by MALDI-TOF. Peptide mass fingerprints from proteins of the 28 spots were obtained by MALDI-TOF mass analysis. The resulting spectra were used to identify the proteins with the Peptident search program. The up- or down-regulated proteins by E7 oncogene are listed in Table 1. Two proteins were downregulated in HaCaT/mock control cells compared to HaCaT/ E7 transfectants (Table 1A). Actin and leukocyte elastase inhibitor were suppressed by E7 expression. Compared to HaCaT/mock control cells, 26 proteins were up-regulated in HaCaT/E7 (Table 1B). Stress-induced phosphoprotein 1 (Hsp70/Hsp90-organizing protein; HOP), CD2 binding protein 1 (proline –serine– threonine phosphatase interacting protein 1), catalase, T-complex protein (TCP) 1, Ku70binding protein (Ku70BP), heat shock 60 kDa protein (hsp60) 1, G1/S-specific cyclin E1 and peroxiredoxin (Prx) 2 were induced by E7 expression. Two-dimensional electrophoresis patterns of E7-interacting proteins from HaCaT cell lysates after transfection with E7 and identification of E7-interacting proteins modulated by E7 expression using mass spectrometry In order to find the factors interacting with E7, the E7affinity column was prepared to obtain E7-interacting proteins. We also compared the differential patterns of E7interacting proteins in 2-D between mock vector and E7transfected cell. Twenty-two protein spots were identified from the selected parts of the 2-D gels. Twenty-two spots were up-regulated and bound to E7 (Fig. 2). Peptide mass fingerprints from 20 proteins of the 22 spots were obtained by MALDI-TOF mass analysis. The up-regulated proteins by E7 oncogene are listed in Table 2. Proteins similar to mouse IFNgamma inducible MG11, livin inhibitor-of-apoptosis, protein serine kinase c17, CD2 binding protein 1, G1/S-specific cyclin E1, TATA box binding protein (TBP)-associated factor (RNA polymerase II) and uridine– cytidine kinase 2 were induced by E7 expression in HaCaT/E7. The levels of proteins down- or up-regulated by E7 In order to confirm the levels of proteins regulated by E7, Western blots were performed in E7-expressing cell lysates

and E7-affinity column binding proteins for 2-DE. Heat shock protein 60 was increased by E7 in accordance with the pattern of 2-DE and MALDI-TOF, whereas HOP and TCP-1 were not significantly influenced by E7 expression (Fig. 3). These results suggest that proteomics approach does not guarantee the correct proteins and thus just identify some candidate proteins when we perform the database search using PEPTIDENT of the program ExPASY (http://cn. expasy.org/tools/peptident.html). In vitro bindings between LEI or Ku70BP and E7 Pull down assays were performed in order to investigate the direct involvement of LEI or Ku70BP with E7. Histagged Ku70BP was also bound to GST-E7 (Fig. 4). The interaction between LEI and E7 was confirmed by binding the LEI-expressing cell lysates into the GST-E7-affinity column. LEI-flag was expressed in HEK293 cells and was used to bind to the E7-affinity column, and LEI was detected by immunoblot (Fig. 5). Discussion Human papillomavirus (HPV) 16 is the most common virus that causes human cervical carcinoma, which is associated with viral oncogenes E6 and E7 of the high risk groups. The present study was performed to investigate the proteins involved in the immune escape and oncogenesis in HPV-induced cervical cancer cells. By using proteomics, we identified various cellular factors that are suppressed or induced by E7 oncogene. In proteomics, cytoplasmic actin and leukocyte elastase inhibitor (LEI) were down-regulated by E7 oncogene (Table 1A). Actin plays an essential role in regulating cell structure during reorganization of cells via specific association with tight junctions [22]. Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins. These components such as actin, microtubules and motors in the organization of post-Golgi trafficking play key roles in apical and basolateral sorting signals, adaptors for basolateral signals and docking and fusion proteins for vesicular trafficking [23]. Neutrophil elastase is a protease that is involved in the tissue destruction and inflammation

148

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

Fig. 2. Two-dimensional electrophoresis patterns of E7-interacting proteins from HaCaT after transfection with E7 or mock vector. The proteins were separated by 2-dimensional electrophoresis and stained with silver nitrate. Identified protein spots are indicated by numbers. Up-regulated proteins by E7 expression are indicated in the right panel (E7-transfected cells). The numbers indicate the position of the up-regulated protein spots in 2-D pattern by E7. The x axis represents pH 3 – 10 immobilized pH gradient for the first dimension, and the y axis represents the molecular weight of standard markers.

that characterize numerous diseases, including hereditary emphysema, chronic obstructive pulmonary disease, cystic fibrosis, adult respiratory distress syndrome, ischemic – reperfusion injury and rheumatoid arthritis [24]. Thus, elastase has been the object of extensive research to develop potent inhibitors that target its destructive and proinflammatory action. Our proteomics data suggested that E7 may be involved in the modulation of elastase activity by down-regulating the LEI expression (Table 1A). Several proteins including peroxiredoxin 2 (Prx 2), catalase,

Ku70BP, HOP, 26S proteosome subunit, TCP-1 and hsp60 were up-regulated by E7 expression (Table 1B). Our recent data also showed that Prx 2 and catalase were increased by E7 expression in Western blot and 2-DE [13]. Prx 2 has been known to be induced by various oxidative stimuli, not only to protect cells from oxidative damage caused by hydrogen peroxide (H2O2), but also to contribute to chemoresistance of cancer cells to H2O2 and anticancer drugs, thereby inhibiting apoptosis [25,26]. Catalase is also known to have anti-oxidant effects on

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

Fig. 3. Western blot analysis of E7-modulating proteins in HaCaT. The proteins modulated by E7 were resolved in SDS-PAGE and transferred onto the PVDF membrane as described in the Materials and methods section.

various oxidative stimuli, such as hydrogen peroxide (H2O2). Recently, it has been reported that overexpression of HPV E7 genes protected the cells from apoptotic death after treatment of H2O2. Astrocytes and keratinocytes infected with E7 oncogene showed higher catalase activity compared with the control cells. These results exhibit an alternative viral escape mechanism exerting E7-transfectants protection from H2O2 injury at least partly due to the increased catalase activity [13,27 – 29]. Chaperone-related proteins such as HOP, TCP-1 and hsp60 were up-regulated in 2-DE. However, hsp60 was only up-regulated, while the levels of HOP and TCP-1 were not significantly influenced by E7 (Fig. 3). Database search using PEPTIDENT of the program ExPASY provides us some pieces of information that can identify some candidate proteins. Among up-regulated spots listed in Table 1B, 3 or 4 candidate proteins were overlapped in several selected

Fig. 4. In vitro binding assay between GST-E7 and his-Ku70BP. In vitro binding assay between GST-E7 and recombinant his-Ku70BP was performed as described in the Materials and methods section. Antibodies to Ku70BP or E7 were used to detect Ku70BP or E7, respectively. Lane 1, his-Ku70BP. Lane 2, control of GST alone mixed with his-Ku70BP. Lane 3, GST-E7 mixed with his-Ku70BP.

149

Fig. 5. In vitro binding assay between GST-E7 and lysates of flag-tagged LEI-transfected HEK 293 cells. In vitro binding assay between GST-E7 and LEI was performed as described in the Materials and methods section. Antibodies to flag or E7 were used to detect LEI (A) or E7 (B), respectively. Lane 1, control of GST alone mixed with LEI-flag transfectants. Lane 2, GST-E7 mixed with LEI-flag transfectants.

spots (#10, #15, etc) from database search. Thus, HOP and TCP-1 might not be up-regulated by E7. HOP is a 60 kDa protein characterized by its ability to bind to two chaperones, hsp70 and hsp90. The molecular chaperones hsp70 and hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. It has been reported that hsp70, hsp90 and Hop comprise a ternary multichaperone complex [30]. Hsp70, a general molecular chaperone, was known to be related to the induction of cellular immune response or various cellular processes, including the transport of protein across membrane. Hsp90 stabilizes a substrate with high affinity. It is known that Hop provides a physical link between hsp70 and hsp90 and also modulates the activities of both of these chaperone proteins. However, HOP does not act independently [31,32]. The cytosolic chaperonin-containing T-complex polypeptide 1 (CCT) is a molecular chaperone that plays an important role in the folding of proteins in eukaryotic cytosol. Actin, tubulin and several other proteins are known to be folded by CCT. The overall expression of CCT in mammalian cells is primarily dependent on cell growth [33]. CCT expression is strongly up-regulated during cell growth, especially from G(1)/S transition to early S phase. CCT is expected to play important roles for growth of cancer cells by assisting the folding of tubulin and other proteins [34]. Hsp60 is already known to be highly expressed in various cancers [35]. It is presumed that hsp60 may interact with a variety of cellular proteins and has a role in cervical carcinogenesis [36,37]. These results indicate that chaperone-related proteins can be directly or indirectly related to oncogenesis by E7. Ku70 and Ku80 are essential for DNA double-strand break repair. It is suggested that the Ku70BP amplification and overexpression may be involved in the function of Ku70 in the DNA –PK complex involved in the maintenance of genome stability and reduction of mutation frequency [38]. Ku70BP was up-regulated by E7 expression in 2-DE and also bound to E7 in in vitro binding assay (Fig. 4). It is assumed that E7-induced up-regulation of Ku70BP may contribute to the functioning of Ku70, which plays roles in double-strand DNA break repair and maintenance of telomeres [39].

150

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

Actin and LEI, which is known as an anti-protease of serpin superfamily, were down-regulated by E7 expression (Table 1A). L-DNase II is involved in apoptosis induced by LEI. It has been recently reported that LEI has a dual role in living and apoptotic cells, suggesting that it prevents the apoptotic cascade in the living cells on its native or stressed conformation, but it digests nuclear DNA after transition to DNase [40]. The interaction between LEI and E7 suggests that apoptotic signal of LEI can be inhibited by E7 (Fig. 5). Livin inhibitor-of-apoptosis, G1/S-specific cyclin E1 and TATA box binding protein (TBP)-associated factor (RNA

polymerase II) were bound to E7 and induced by E7 expression in HaCaT/E7 (Table 2). Livin inhibitor-ofapoptosis is a novel human inhibitor of apoptosis protein (IAP) family member termed livin. The mRNA for livin was not detectable in most normal adult tissues, but it was present in developmental tissues and in several cancer cell lines. Livin was expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. Disruption of livin may provide a strategy to induce apoptosis in certain cancer cells [41]. Prospectively, cyclin E1 was up-regulated by E7. Cyclin E controls the initiation of DNA synthesis by activating CDK2. In addition, abnormally high level of cyclin E

Table 2 Identification of E7-interacting proteins induced by E7 expression using mass spectrometry Spot

Score

# peptide matches

AC

Description

pI

Mw

Arbitrary intensity

3

0.14 0.11 0.12

5 4 4

Q9Y3Z8 Q9Y3A3 O95541

Undefined 5.5 Undefined

Undefined 26,032.43 Undefined

+

0.15

4

Q9H004

Undefined

Undefined

+ ++ ++

7

0.14

4

Q9HAP7

5.52

30,865.77

+++

8 9 10

0.14 0.11 0.12 0.12 0.12 0.12 0.13 0.22 0.22 0.23 0.25

4 4 4 4 4 4 4 4 4 5 5

9.12 Undefined 9.32 Undefined Undefined 8.84 Undefined 5.36 5.49 6.11 5.58

17,624.15 Undefined 21,581.81 Undefined Undefined 24,819.04 Undefined 52,964.94 43,247.56 54,409.22 58,946.3

++ ++ ++

0.2 0.15 0.15 0.15

4 4 4 4

Q9Y3D9 Q9UM79 Q96HP7 Q9UFU2 Q9UGL7 Q9Y2R6 Q92531 O94948 Q9UI00 Q92943 P98082 (isoform) Q96HX7 P49411 Q96SD6 Q9NV76

Undefined 6.31 Undefined 5.42

Undefined 45,045 Undefined 58,158.01

16

0.19

4

Q13426

4.91

38,057.51

+

17

0.14

4

O43586

5.35

47,591.36

++

18

0.11 0.11

4 4

P24864 O43586

5.7 5.35

47,077.19 47,591.36

+

0.11 0.16 0.12 0.12 0.12 0.17

4 5 4 4 4 4

P36955 O43585 P30532 Q9BUU9 Q9UP07 P17174

5.84 5.28 6.04 Undefined Undefined 6.57

44,819.28 45,353.74 50,809.71 Undefined Undefined 46,116.31

0.17

4

Q9BR40

Undefined

Undefined

0.18 0.12 0.12

4 4 4

Q9Y3Z8 Q95J02 Q96KG5

Hypothetical 33.3 kDa protein (fragment) CGI-95 PROTEIN (MOB3) (unknown) (protein for mGC:12264) DJ283E3.2.3 (matrix metalloproteinase MMP21/22B) (fragment) No SWISS-PROT and TrEMBL entries have been found DJ132F21.3 (72.1 kDa protein (DKFZP564A032, SBBI88) similar to mouse IFN-gamma induce MG11) (fragment) Livin inhibitor-of-apoptosis (inhibitor of apoptosis) (BA261N11.1.1) (baculoviral IAP repeat-containing protein 7 (livin), isoform 1) Hypothetical protein CGI-138 Protein serine kinase c17 (fragment) DKFZP586G1722 protein Hypothetical 23.3 kDa protein (fragment) NICE-3 protein (fragment) HSPC012 (DKFZP586G1722 protein) P35-related protein (fragment) KIAA0871 protein Rap2 interacting protein x Farnesol receptor HRR-1 SPLICE isoform 2 of disabled homolog 2 (differentially expressed protein 2) (DOC-2) Similar to heat shock 90 kDa protein 1, alpha (fragment) CHAIN 1: ELONGATION FACTOR TU Putative TCPTP-interacting protein (fragment) cDNA FLJ10887 fis, clone NT2RP4002018, weakly similar to RING CANAL PROTEIN DNA DOUBLE-STRAND BREAK REPAIR AND V(D)J RECOMBINATION PROTEIN XRCC4 CD2 binding protein 1 long form (proline – serine – threonine phosphatase interacting protein 1) G1/S-specific cyclin E1 CD2 binding protein 1 long form (proline – serine – threonine phosphatase interacting protein 1) CHAIN 1: PIGMENT EPITHELIUM-DERIVED FACTOR CD2 binding protein 1 short form CHAIN 1: NEURONAL ACETYLCHOLINE RECEPTOR PROTEIN Similar to tubulin, beta 5 (fragment) Cytochrome P450 21-hydroxylase (fragment) Aspartate aminotransferase, cytoplasmic (EC 2.6.1.1) (transaminase A) (glutamate oxaloacetate transaminase-1) DJ1107C24.1 (TATA box binding protein (TBP)-associated factor, RNA polymerase II, C1, 130 kDa) (fragment) Hypothetical 33.3 kDa protein (fragment) MHC class I antigen (fragment) Uridine – cytidine kinase 2 (fragment)

Undefined Undefined Undefined

Undefined Undefined Undefined

4 5 6

11 12 13 14

15

19

20

21 22

++ ++ ++ +

+

++

++

++ ++

Up-regulated proteins by E7 are described. The peptide profiles of the protein spots treated with trypsin were analyzed by MALDI-TOF-MS and by using the Expasy Peptident search program.

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152

expression has frequently been observed in human cancers. The expression of these two G1 E-type cyclins may be similarly regulated by the pRb function, but distinctly regulated by the p53 activity [42]. E7 is capable of interacting with various cellular protein targets, including RB105, TATA box-binding protein (TBP), TBP-associated factor (TAF) (II) 110, E2F, cyclins A and D and c-jun [43]. TBP-associated factor was up-regulated by E7 expression and was bound to E7. It is known that E7 binds to 921 amino acids of TBP-associated factor-110 (TAF-110) as well as TBP. E7 can activate promoters through protein interactions with components of the transcription inhibition complex, and it is expected that E7 may modulate the expression of specific promoters, which could contribute to the pathogenesis of human papillomavirus [44]. Up-regulated proteins by E7 in HaCaT/E7 were corresponded with proteins identified in C33A/E7. 26S proteosome subunit, Ku70BP and hsp60 were also upregulated by C33A/E7 [45]. Our results showed that all Western blot data were not always correlated with proteomics results. However, these results suggest that the infection of E7 oncogene into epithelial cells can evade immune surveillance or resist against apoptosis by inducing or binding to various factors such as chaperone, cell signaling factors and cell cycle regulatory factors. This study also provides a new and useful information for understanding the alternative viral escape mechanisms of HPV 16 E7-infected keratinocytes.

[6]

[7]

[8]

[9]

[10] [11] [12]

[13]

[14]

[15]

[16]

Acknowledgments This work was supported by a grant from KRIBB Research Initiative Program and a grant (PF0321001-00) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by Ministry of Science and Technology and by grant No. RT104-03-07 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE). The authors thank Ms. So-Young Kim for her critical proofreading.

[17]

[18]

[19]

[20]

References [1] zur Hausen H, de Villiers EM. Human papillomaviruses. Annu Rev Microbiol 1994;48:427 – 47. [2] Filatov L, Golubovskaya V, Hurt JC, Byrd LL, Phillips JM, Kaufmann WK. Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein. Oncogene 1998;16:1825 – 38. [3] Syrjanen SM, Syrjanen KJ. New concepts on the role of human papillomavirus in cell cycle regulation. Ann Med 1999;31:175 – 87. [4] Zwerschke W, Jansen-Durr P. Cell transformation by the E7 oncoprotein of human papillomavirus type 16: interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res 2000;78:1 – 29. [5] Clark PR, Roberts ML, Cowsert LM. A novel drug screening assay for

[21]

[22]

[23]

[24]

[25]

151

papillomavirus specific antiviral activity. Antiviral Res 1998;37: 97 – 106. Scheffner M, Romanczuk H, Munger K, Huibregtse JM, Mietz JA, Howley PM. Functions of human papillomavirus proteins. Curr Top Microbiol Immunol 1994;186:83 – 99. Lee JE, Kim CY, Giaccia AJ, Giffard RG. The E6 and E7 genes of human papillomavirus-type 16 protect primary astrocyte cultures from injury. Brain Res 1998;795:10 – 6. Phillips AC, Vousden KH. Analysis of the interaction between human papillomavirus type 16 E7 and the TATA-binding protein, TBP. J Gen Virol 1997;78:905 – 9. Antinore MJ, Birrer MJ, Patel D, Nader L, McCance DJ. The human papillomavirus type 16 E7 gene product interacts with and transactivates the AP1 family of transcription factors. EMBO J 1996;15: 1950 – 60. Lockhart DJ, Winzeler EA. Genomics, gene expression and DNA arrays. Nature 2000;405:827 – 36. Pandey A, Mann M. Proteomics to study genes and genomes. Nature 2000;405:837 – 46. Le Naour F, Hohenkirk L, Grolleau A, Misek DE, Lescure P, Geiger JD, et al. Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J Biol Chem 2001;276: 17920 – 31. Shim JH, Cho KJ, Lee KA, Kim SH, Myung PK, Choe YK, et al. E7expressing HaCaT keratinocyte cells are resistant to oxidative stressinduced cell death via the induction of catalase. Proteomics 2005;5: 2112 – 22. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate – phenol – chloroform extraction. Anal Biochem 1987;162:156 – 9. Cho Y, Cho C, Joung O, Lee K, Shim J, Park S, et al. Development of screening systems for drugs against human papillomavirus-associated cervical cancer: based on E7-Rb binding. J Biochem Mol Biol 2001;34:80 – 4. Cho YS, Kang JW, Cho M, Cho CW, Lee S, Choe YK, et al. Down modulation of IL-18 expression by human papillomavirus type 16 E6 oncogene via binding to IL-18. FEBS Lett 2001;501:139 – 45. Lee SJ, Cho YS, Cho MC, Shim JH, Lee KA, Ko KK, et al. Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18induced IFN-gamma production in human peripheral blood mononuclear and NK cells. J Immunol 2001;167:497 – 504. Strahler JR, Kuick R, Hanash SM. Diminished phosphorylation of a heat shock protein (HSP 27) in infant acute lymphoblastic leukemia. Biochem Biophys Res Commun 1991;175:134 – 42. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20:601 – 5. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996;68:850 – 8. Gharahdaghi F, Kirchner M, Fernandez J, Mische SM. Peptide-mass profiles of polyvinylidene difluoride-bound proteins by matrixassisted laser desorption/ionization time-of-flight mass spectrometry in the presence of nonionic detergents. Anal Biochem 1996;233: 94 – 9. Sjo A, Magnusson KE, Peterson KH. Association of alphadystrobrevin with reorganizing tight junctions. J Membr Biol 2005; 203:21 – 30. Rodriguez-Boulan E, Kreitze G, Musch A. Organization of vesicular trafficking in epithelia. Nat Rev, Mol Cell Biol 2005;6: 233 – 47. Tremblay GM, Janelle MF, Bourbonnais Y. Anti-inflammatory activity of neutrophil elastase inhibitors. Curr Opin Invest Drugs 2003;4:556 – 65. Yo YD, Chung YM, Park JK, Ahn CM, Kim SK, Kim HJ. Synergistic

152

[26]

[27]

[28]

[29]

[30] [31]

[32] [33] [34]

[35]

K.-A. Lee et al. / Gynecologic Oncology 99 (2005) 142 – 152 effect of peroxiredoxin II antisense on cisplatin-induced cell death. Exp Mol Med 2002;34:273 – 7. Chung YM, Yoo YD, Park JK, Kim YT, Kim HJ. Increased expression of peroxiredoxin II confers resistance to cisplatin. Anticancer Res 2001;21:1129 – 33. Lee WT, Lee JE, Lee SH, Jang HS, Giffard RG, Park KA. Human papillomavirus type 16 E7 genes protect astrocytes against apoptotic and necrotic death induced by hydrogen peroxide. Yonsei Med J 2001;42:471 – 9. Alayash AI, Ryan BA, Eich RF, Olson JS, Cashon RE. Reactions of sperm whale myoglobin with hydrogen peroxide. Effects of distal pocket mutations on the formation and stability of the ferryl intermediate. J Biol Chem 1999;274:2029 – 37. Brantley Jr RE, Smerdon SJ, Wilkinson AJ, Singleton EW, Olson JS. The mechanism of autooxidation of myoglobin. J Biol Chem 1993;268:6995 – 7010. Wegele H, Haslbeck M, Reinstein J, Buchner J. Sti1 is a novel activator of the Ssa proteins. J Biol Chem 2003;278:25970 – 6. Johnson BD, Schumacher RJ, Ross ED, Toft DO. Hop modulates Hsp70/Hsp90 interactions in protein folding. J Biol Chem 1998; 273:3679 – 86. Wegele H, Muller L, Buchner J. Hsp70 and Hsp90—A relay team for protein folding. Rev Physiol Biochem Pharmacol 2004;151:1 – 44. Kubota H. Function and regulation of cytosolic molecular chaperone CCT. Vitam Horm 2002;65:313 – 31. Yokota S, Yanagi H, Yura T, Kubota H. Cytosolic chaperonin is upregulated during cell growth. Preferential expression and binding to tubulin at G(1)/S transition through early S phase. J Biol Chem 1999;274:37070 – 8. Kimura E, Enns RE, Thiebaut F, Howell SB. Regulation of HSP60 mRNA expression in a human ovarian carcinoma cell line. Cancer Chemother Pharmacol 1993;32:279 – 85.

[36] Cappello F, Bellafiore M, Palma A, Marciano V, Martorana G, Belfiore P, et al. Expression of 60-kD heat shock protein increases during carcinogenesis in the uterine exocervix. Pathobiology 2002;70:83 – 8. [37] Garrido C, Gurbuxani S, Ravagnan L, Kroemer G. Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 2001;286:433 – 42. [38] Leskov KS, Klokov DY, Li J, Kinsella TJ, Boothman DA. Synthesis and functional analyses of nuclear clusterin, a cell death protein. J Biol Chem 2003;278:11590 – 600. [39] Song K, Jung D, Jung Y, Lee SG, Lee I. Interaction of human Ku70 with TRF2. FEBS Lett 2000;481:81 – 5. [40] Perani P, Zeggai S, Torriglia A, Courtois Y. Mutations on the hinge region of leukocyte elastase inhibitor determine the loss of inhibitory function. Biochem Biophys Res Commun 2000;274:841 – 4. [41] Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 2001;276:3238 – 46. [42] Kinjo T, Kamiyama K, Chinen K, Iwamasa T, Kurihara K, Hamada T. Squamous metaplasia induced by transfection of human papillomavirus DNA into cultured adenocarcinoma cells. Mol Pathol 2003;56: 97 – 108. [43] Hermonat PL, Santin AD, Zhan D. Binding of the human papillomavirus type 16 E7 oncoprotein and the adeno-associated virus Rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects. J Hum Virol 2000;3:113 – 24. [44] Mazzarelli JM, Atkins GB, Geisberg JV, Ricciardi RP. The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40 TAg bind a common region of the TBP-associated factor-110. Oncogene 1995;11:1859 – 64. [45] Lee K, Shim J, Kho C, Park S, Park B, Kim J, et al. Protein profiling and identification of modulators regulated by the E7 oncogene in the C33A cell line by proteomics and genomics. Proteomics 2004;4: 839 – 48.