Tumor Suppressor Gene 14-3-3 Is Down-Regulated whereas the

Proto-Oncogene Translation Elongation Factor 1δ Is Up-Regulated in ... It has been suggested that molecular changes including overexpression of oncog...
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Tumor Suppressor Gene 14-3-3σ Is Down-Regulated whereas the Proto-Oncogene Translation Elongation Factor 1δ Is Up-Regulated in Non-Small Cell Lung Cancers As Identified by Proteomic Profiling Yang Liu, Qun Chen, and Jian-Ting Zhang* Department of Pharmacology and Toxicology, Walther Oncology Center/Walther Cancer Institute and IU Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 Received December 18, 2003

Lung cancer, a leading cause of cancer deaths, consists of two major groups: small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC) with the NSCLC accounting for ∼75% cases of lung cancers. It has been suggested that molecular changes including overexpression of oncogenes and decreased expression of tumor suppressor genes are responsible for lung carcinogenesis. In this study, we analyzed protein profiles of four different human NSCLC cell lines compared with normal human bronchial epithelial cells using two-dimensional PAGE and MALDI-TOF mass spectrometry. We identified 12 protein spots with different expressions between the normal and cancer cells. Of these proteins, vimentin, cytokeratin 8, YB-1, PCNA, Nm23, hnRNP A2/B1, and HSP90β were known to be up-regulated in lung cancers, which is consistent with the current study. We also found that the expression of M-type pyruvate kinase is altered in NSCLC likely due to changes in translational control and/or differential phosphorylation of the protein. Interestingly, the expression of the tumor suppressor gene 14-3-3σ is down-regulated while that of the proto-oncogene TEF1δ is up-regulated in NSCLC cells. On the basis of these observations and previous studies, we propose that the altered expression of 14-3-3σ and TEF1δ may be involved in lung carcinogenesis. Keywords: proteomics • lung cancer • 14-3-3σ • TEF1δ • pyruvate kinase • MALDI-TOF

Introduction Lung cancer is a malignant disease of heterogeneous histologies and has been divided into two major groups: small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC). NSCLC, accounting for 75% of lung cancers, falls into three major types: squamous cell carcinoma (SCC), adenocarcinoma, large cell lung cancer (LCLC), and infrequently found carcinoids.1 Lung cancer is a leading cause for cancer deaths in both male and female populations (American Cancer Society, Cancer Facts and Figures, 2002). The number of deaths due to lung cancer approximates to the deaths of breast, prostate, colorectal, and pancreatic cancers combined in 2002. Clearly, studies on lung carcinogenesis are urgently needed and the finding of new diagnostic markers and targets of treatment for lung cancers will help reduce the lung cancer mortality rate. The accumulating evidence suggests that molecular changes including activation or overexpression of oncogenes and inactivation or decreased expression of tumor suppressor genes are responsible for lung carcinogenesis.2 Genetic changes such as hypermethylation of p16, p53 mutation, and deletion of chromosome 3p, 9p, and 17p were frequently detected in lung * To whom correspondence should be addressed: IU Cancer Center, Indiana University School of Medicine, 1044 W. Walnut Street, R4-166, Indianapolis, Indiana 46202. Tel: (317) 278-4503. Fax: (317) 274-8046. E-mail [email protected].

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Journal of Proteome Research 2004, 3, 728-735

Published on Web 06/02/2004

hyperplasia and dysplasia.3-7 Carcinogens in the smoke of cigarettes are widely accepted to be the culprit for inducing multiple genetic and epigenetic alterations. Many of the tumor suppressor genes and oncogenes that were found altered in lung cancers are involved in regulating cell cycle progression and cell growth. In this study, we analyzed protein profile of four human NSCLC cell lines in comparison with one immortalized human normal bronchial epithelial cell line and primary normal human bronchial epithelial cells using two-dimensional PAGE in combination with MALDI-TOF mass spectrometry. Several known protein markers that are overexpressed in lung cancers were identified. Interestingly, we also found that the level of tumor suppressor 14-3-3σ was drastically down-regulated whereas the level of a proto-oncogene translation elongation factor 1δ was increased in all four NSCLC cell lines. These results suggest that the altered expression of 14-3-3σ and translation elongation factor 1δ possibly relate to nonsmall cell lung carcinogenesis.

Experimental Procedures Materials. All electrophoresis reagents, precast criterion SDSPAGE slab gels, immobilized pH gradient (IPG) strips, iodoacetamide, and PVDF membranes were purchased from BioRad. Dithiothreitol (DTT), acetonitrile, and R-cyano-4-hydroxycinnamic acid were obtained from Sigma. Modified trypsin was 10.1021/pr034127+ CCC: $27.50

 2004 American Chemical Society

research articles

Proteomic Analysis of NSCLC Cell Lines

purchased from Promega. Antibody N-14 against 14-3-3σ was purchased from Santa Cruz Biotechnology. SYBR Green PCR Master Mix for real time PCR was purchased from Applied Biosystems. RPMI1640, BEGM (bronchial epithelial growth media), and SFM supplement (human recombinant EGF and bovine pituitary extract) were purchased from Cambrex, whereas R-ΜΕΜ and keratinocyte SFM were from Invitrogen. Human lung cancer cell lines H1299, H23, H226, SW-1573, and the immortalized human normal bronchial epithelial cell line HBE4-E6/E7 were purchased from ATCC. The primary human normal bronchial epithelial cells NHBE-8917 were purchased from Cambrex. Cell Culture and Sample Preparation for 2-DE. H1299, H23, and H226 cells were cultured in RPMI1640, whereas SW1573 was cultured at 37 °C in R-MEM both supplemented with 10% fetal bovine serum. HBE4-E6/E7 was cultured in keratinocyteSFM medium supplemented with human recombinant EGF and bovine pituitary extract at final concentrations of 5 ng/ mL and 50 µg/mL, respectively, while NHBE-8917 was cultured in BEGM. After the cells grow to near confluency in 100 mm dishes, they were rinsed with PBS and lysed in lysis buffer (1% Triton X-100, l50mM NaCl, 10mM Tris pH 7.4, 1mM EDTA, 1mM EGTA pH 8.0, 0.2mM sodium ortho-vanadate, 0.2mM PMSF, 0.5% NP-40) for 30 min at 4 °C with constant agitation. The cell lysates were then collected using a rubber policeman and homogenized by passing through a 26 gauge needle several times followed by centrifugation (16 000 × g, 4 °C) for 15 min to remove insoluble materials. The protein concentration of supernatants from all cell lysates were measured by Bradford assay.8 Two-Dimensional Gel Electrophoresis (2-DE). Two hundred µg each of the six cell lysates were first diluted to 2 µg/µL with lysis buffer and precipitated by acetone and the proteins were collected by centrifugation. The protein pellets were washed once with ice-cold acetone followed by solubilization in 180 µL BioRad rehydration buffer (8 M urea, 50 mM DTT, 4% CHAPS, 0.2% carrier ampholytes [3-10], 0.0001% bromophenol blue) with brief sonication and centrifugation. Each sample was then loaded onto one of the six IPG strips (pH3-10) by passive absorption overnight for the first dimensional run using the Bio-Rad PROTEAN IEF Cell. The IPG strips were isoelectrically focused using a 3-step program (linear 250 V for 20 min; linear 8000 V for 2.5 h; rapid 8000 V for 20 000 V hours). The strips were then treated with reducing buffer (2% DTT, 6 M urea, 2% SDS, 20% glycerol, and 0.375 M Tris/HCl, pH8.8) for 10 min with agitation followed by alkylation with 2.5% iodoacetamide in the same buffer but without DTT for 10 min. The IPG strips were then washed with the SDS-PAGE running buffer and placed into BioRad precast 4-15% gradient gels and covered with Bio-Rad’s ReadyPrep overlay agarose followed by electrophoresis at 130 V. Following the second-dimension run, the gels were stained with Coomassie staining solution (30% 2-propanol, 7% acetic acid, 0.25% coomassie brilliant blue R250) for overnight. The 2-DE gels were then destained using the destaining solution (18% methanol, 5% acetic acid). Image Analysis, MALDI-TOF Mass Spectrometry, and Database Search. The image analysis, MALDI-TOF mass spectrometry, and database search were performed essentially the same as previously described.9 Briefly, the images of the 2-DE gels were scanned and analyzed using Fluor-S MAX MultiImager system and PD Quest software (Bio-Rad) following destaining. The quantity of each spot on the 2-DE gels was defined as parts-per-million (PPM) of the total integrated optical density.

The abundance of the individual protein was calculated using a quantitative analysis set of the PD Quest software. Protein spots of interest were then excised from the gels which have the highest level of the protein, and processed robotically using the PROTEAN 2D Spot Cutter (Bio-Rad) and the MassPREP Workstation (Perkin-Elmer), respectively. Briefly, the excised spots (gels) were placed in a 96-well plate and destained with 50% acetonitrile/50 mM ammonium bicarbonate, reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide followed by digestion with 6 ng/µL trypsin for overnight at 37 °C. R-cyano-4-hydroxycinnamic acid was used as matrix. MALDI (matrix-assisted laser desorption/ionization) mass spectra were recorded in positive reflectron mode of the MALDI-TOF mass spectrometer (Micromass). The time-of-flight (TOF) was measured using 3400 V pulse voltage, 15000 V source voltage, 500 V reflectron voltage, 1950 V MCP voltage, and low mass gate of 500 Da. Internal calibration was performed using auto digestion peaks of bovine trypsin (M+H+, m/z 842.5099 and m/z 2211.1045). The peptide matching and protein searching were performed using ProFound search engine and the NCBI database. Western Blot Analysis. Western blot analysis was performed as previously described.10 Briefly, cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. The blot was then probed with affinity-purified polyclonal 14-3-3σ antibody N-14 (1:100 dilution), followed by reaction with HRPconjugated secondary antibody. The signal was captured by X-ray films using enhanced chemluminensence (ECL). The protein level was determined using a gel densitometer and analyzed using the Scion image software. Real-Time Quantitative PCR. Total RNAs were isolated from both normal and cancer cells by RNeasy mini kit according to the manufacturer’s instruction (Qiagen) and treated with RQ1 RNase-free DNase I. Four µg total RNAs from each cell type were reverse transcribed using AMV Reverse Transcriptase and Oligo(dT)12-18 primer (Invitrogen). Primers for real time PCR were designed using Primer Express software version 2.0 (Applied Biosystems) and synthesized by Invitrogen. The sequences of primers used are as follows: pyruvate kinase M1: forward, 5′-AGTCACTCCACAGACCTCATGGA-3′, reverse, 5′- TCAAAGCTGCTGCTAAACACTTATAAG-3′, pyruvate kinase M2: forward, 5′-CCACTTGCAATTATTTGAGGAACTC-3′, reverse, 5′-ACGGCGGTGGCTTCTGT-3′, TEF1δ: forward, 5′-CCTGCACTGGTGGCCAAGTC-3′, reverse, 5′-AGCTGGATAGAGCGCACACA-3′; 14-3-3σ: forward, 5′-GGCCATGGACATCAGCAAGAA3′, reverse, 5′-CGAAAGTGGTCTTGGCCAGAG-3′; β-actin: forward, 5′-CCGACAGGATGCAGAAGGA-3′, reverse, 5′- TCAGGAGGAGCAATGATCTTGAT-3′. Real time quantitative PCR was carried out in ABI Prism@7000 Sequence Detection System (Applied Biosystems) using SYBR Green according to the manufacturer’s instruction. The threshold cycle (Ct) was defined as the PCR cycle number at which the reporter fluorescence crosses the threshold reflecting a statistically significant point above the calculated baseline. The Ct of each target product was determined, and normalized against that of the housekeeping gene β-actin. Fold difference ) 2∆Ct.

Results Total cell lysates were prepared from human NSCLC cell lines H1299 (large cell carcinoma), SW1573 (alveolar cell carcinoma), H23 (adenocarcinoma), H226 (squamous cell carcinoma), and from immortalized normal human bronchial epithelial cell line HBE4-E6/E7 and primary normal human Journal of Proteome Research • Vol. 3, No. 4, 2004 729

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Figure 1. Two-dimensional gel electrophoresis of NSCLC and normal cell lysates. Two hundred µg proteins of H1299, SW1573, H23, H226, HBE4-E6/E7, and NHBE cell lysates were first separated by IEF (pH 3-10) followed by SDS-PAGE (4-15% gradient) and stained with commassie blue. The protein profile was analyzed using PD Quest software (Bio-Rad). Each panel represents one of the triplicate runs. The protein spots of differential expression between normal and cancer cells and identified by MALDI-TOF are indicated by arrows.

bronchial epithelial (NHBE) cells and then subjected to twodimensional gel electrophoresis analysis (Figure 1). Protein spots that have consistent differences (>2-fold) between all four NSCLC cell lines and the normal cells in all three separate experiments were chosen for further analysis. Forty-eight such spots were found when analyzed using PD Quest imaging system and were subjected to MALDI-TOF mass spectrometry analysis. Thirty-three of these spots generated quality mass spectrometry data that can be used reliably to identify the proteins. However, 21 of these proteins that were found exclusively in NSCLC cell lines were identified to be contaminating proteins from bovine serum which were used for culturing the cancer but not the normal cells (data not shown). The remaining 12 spots (Figure 1, Figure 2 and Table 1) were identified by MALDI-TOF with good peptide coverage and reasonable Z-scores (Table 2). Except YB-1 of which the observed pI is different from the calculated pI, all other proteins have similar observed pI and molecular weight to the calculated ones. Of the 12 cellular proteins identified, most of them (vimentin, cytokeratin 8, YB-1, PCNA, Nm23, hnRNP A2/B1, and HSP90β) 730

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are known lung cancer markers or have been shown to have an altered expression in lung cancers which are consistent with the present study (Table 3). Of particular interest, however, is the tumor suppressor 14-3-3σ (also called stratifin) which was found to be decreased in NSCLC cell lines. To verify this finding, we performed real time RT-PCR and western blot analysis to detect the expression level of mRNA and protein, respectively. As shown in Figure 3, the expression of 14-3-3σ at both mRNA (Figure 3A) and protein (Figure 3B) levels was decreased in all four NSCLC cell lines compared with normal cells. This observation is consistent with that generated by PD Quest analysis of the 2-DE gels (see Table 1). Thus, 14-3-3σ expression is down-regulated in NSCLC cell lines H1299, SW1573, H23, and H226 compared with normal bronchial epithelial cells. Another spot of interest is the proto-oncogene translation elongation factor 1δ (TEF1δ, Table 2) and no studies on its relationship with lung cancers have been reported previously. To verify that its expression is increased in NSCLC cell lines, we performed only a real-time PCR analysis because of the lack of antibody for this protein. As shown in Figure 4, the mRNA level of TEF1δ is higher in cancer cells than that in normal cells,

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Proteomic Analysis of NSCLC Cell Lines

Figure 2. Master image of the 2-DE gel profile of 12 proteins identified by MALDI mass spectrometry and peptide mass fingerprinting. The master image was created from the images of all gels (triplicate of all six samples) by the PD Quest program. The proteins that are differentially expressed between NSCLC and normal cells and identified by MALDI-TOF are indicated. Table 1. Relative Quantity of the Protein Spots Identified by MALDI-TOF average relative quantitya protein

H1299

SW1573

H23

H226

HBE4

NHBE

YB-1 14-3-3σ PCNA TEF1δ vimentin cytokeratin 8 HSP90β Nm23 hnRNP A2/B1 M-type pyruvate kinase (A) M-type pyruvate kinase (B) M-type pyruvate kinase (C)

4 0.01 9 56 17 24 3 4 4 0.3

21 0.2 16 38 29 42 3 7 5 0.3

17 0.03 11 77 13 52 3 7 3 0.4

19 0.07 13 59 10 68 3 5 5 0.7

1 0.5 6 48 7 1 2 4 1 0.9

1 1 1 1 1 1 1 1 1 1

5

4

3

7

3

1

0.1

0.1

0.3

0.4

1

1

a

The average relative quantity of each spot was calculated by normalizing the average quantity PPM (parts-per-million measured using PD Quest) of the total integrated optical density of each spot in triplicate gels of each cell line against that of NHBE.

consistent with that generated by PD Quest analysis of the 2-DE gels (see Table 1). Thus, the expression of TEF1δ is likely increased in NSCLC cell lines compared with the normal cells. Three other spots of interest with the same molecular mass but different pI were all identified to be M-type pyruvate kinase (Table 2). While two of them (spots A and C) decreased in all cancer cell lines, the third one (spot B) increased when compared with normal cells (Table 1 and Figure 5). It is known that the M-type pyruvate kinase exists in two isoforms, M1 and M2, a result of alternative mRNA splicing with exons 9 and 10 being mutually exclusive. Mature mRNAs containing exon 9 encodes for the pyruvate kinase M1 whereas mRNAs containing exon 10 encodes for M2. To determine whether these spots are isoform specific, we manually examined the peptide mass fingerprints of these spots and searched for the peptides from MALDI-TOF corresponding to the sequence encoded by exon 9 and 10. We found that while the spots A and C represent isoform M1 the spot B likely consists of both isoform M1 and M2 (data not shown).

To confirm the above findings, we performed real time RTPCR using two pairs of primers targeting specifically to isoform M1 and M2. As shown in Figure 6, we found that the mRNAs of the M1 isoform was decreased in three of the four cancer cell lines compared with the normal cells (Figure 6A), which confirms the findings from the 2-DE and peptide mass fingerprints that the isoform M1 (spots A and C) is decreased in lung cancer cell lines (Table 1). Interestingly, the mRNAs of the M2 isoform did not appear to increase in cancer cells (Figure 6B), inconsistent with our 2-DE and peptide mass fingerprint data, suggesting that the expression of M2 isoform may be altered at translational or posttranslational levels. We also found that the mRNAs of both M1 and M2 isoforms appeared to be higher in the H226 cell line compared to other cancer cell lines, consistent with the 2-DE data (see Table 1).

Discussion In this study, we used proteomic profiling to compare human nonsmall cell lung cancer and normal bronchial epithelial cells and identified 12 protein spots that were up- or down-regulated in four human NSCLC cell lines. Most of these proteins (vimentin, cytokeratin 8, YB-1, PCNA, Nm23, hnRNP A2/B1, and HSP90β) are known lung cancer markers or known to be up-regulated in lung cancers which are consistent with the findings of this study. However, the finding of changes in the expression of three proteins, 14-3-3σ, TEF1δ, and M-type pyruvate kinase is novel and their altered expression in NSCLC cell lines may have implications in lung tumorigenesis and may be used as new diagnostic markers of NSCLC. TEF1δ was found changed between normal and NSCLC cell lines. While no study is available regarding the expression of TEF1δ and human lung cancers, TEF1δ has been shown to be a cadmium-responsive proto-oncogene and causes tumorigenic growth of NIH3T3 cells when overexpressed.11 Blocking the overexpression of TEF1δ by antisense sequences reversed the oncogenic growth of NIH3T3 cells. Thus, it is possible that overexpression of the proto-oncogene TEF1δ may relate to NSCLC. 14-3-3σ, also called stratifin, was found decreased in all four NSCLC cell lines compared to normal bronchial epithelial cells. Journal of Proteome Research • Vol. 3, No. 4, 2004 731

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Table 2. Summary of MALDI-TOF Data of the Identified Protein Spots protein

accession no.a

MW (kDa) (Cb/Oc)

pI (C/O)

coveraged (%)

Z-scoree

YB-1 14-3-3σ PCNA TEF1δ vimentin cytokeratin 8 HSP90β Nm23 hnRNP A2/B1 M-type pyruvate kinase (A) M-type pyruvate kinase (B) M-type pyruvate kinase (C)

P16991 NP_006133 NP_002583 NP_001951 XP_042950 P05787 P08238 CAA35621 NP_112533 A33983 A33983 A33983

36/48 28/28 29/31 31/35 54/44 54/51 84/76 21/26 38/36 59/63 59/63 59/63

9.9/3.7 4.7/4.8 4.6/4.8 4.9/5.2 5.1/5.0 5.5/6.1 5.0/6.0 7.1/6.5 9.0/8.8 8.4/8.0 8.4/8.4 8.4/8.6

35 58 39 37 50 50 37 49 42 33 44 33

2.23 2.40 1.97 2.16 2.35 2.35 1.67 2.19 2.29 2.30 2.34 2.30

a Accesion no. used is from entries in Swiss-Prot. b C)calculated value from the sequence. c O)observed value based on estimation from the two-dimensional gel. d Coverage is defined as the ratio of the portion of protein sequence covered by matched peptides to the whole length of protein sequence. e The Z score is calculated by the ProFound search engine and is an indicator of search result quality. Z score is estimated when the search result is compared against an estimated random match population. Z score is the distance to the population mean in unit of standard deviation. It also corresponds to the percentile of the search in the random match population. A Z score of 1.65 for a search means that the search is in the 95th percentile. In other words, there are about 5% of random matches that could yield higher Z scores than this search.

Table 3. Summary of Correlations between the Expression of the Protein Spots Identified and Human Lung Cancer protein

expressions (this study)

expressions (literature)

14-3-3σ TEF1δ vimentin cytokeratin 8 YB-1 PCNA Nm23 hnRNP A2/B1 pyruvate kinase M1 pyruvate kinase M2 ΗSP90β

low in cancer cells high in cancer cells high in cancer cells high in cancer cells high in cancer cells high in cancer cells high in cancer cells high in cancer cells low in cancer cells high in cancer cells high in cancer cells

low in SCLC no report high in lung cancer high in lung cancer high in lung cancer high in lung cancer high in lung cancer high in lung cancer no report high in lung cancer high in lung cancer

Figure 3. Analysis of 14-3-3σ expression by real time quantitative RT-PCR and western blot. A. Relative mRNA levels of 14-3-3σ in NSCLC and normal cells. Real time quantitative RT-PCR was performed as described in Experimental Methods. Relative mRNA levels were measured using SYBR Green and calculated in the fold change (2∆Ct) relative to NHBE cells after normalization by the internal control, β-actin. B. Relative protein levels of 143-3σ in NSCLC and normal cells. Twenty µg of cell lysates were separated by SDS-PAGE followed by Western blot using 14-33σ antibody as a probe as described in Experimental Procedures. Actin was used as a loading control.

14-3-3σ was found initially as an epithelial cell marker.12 In human, there are seven types of 14-3-3 proteins which are abundant and thought to perform various functions. 14-3-3 proteins can interact with more than 100 cellular proteins at 732

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Figure 4. Analysis of TEF-1δ expression by real time quantitative RT-PCR. Real time quantitative RT-PCR was performed as described in Experimental Procedures. Relative mRNA levels were measured using SYBR Green and calculated in the fold change (2∆Ct) relative to NHBE cells after normalization by the internal control, β-actin.

Figure 5. Two-dimensional gel images of M-type pyruvate kinase in different cells. Three protein spots (indicated by arrows and letters), all identified to be M-type pyruvate kinase, are shown from the original images in Figure 1.

their phosphorylation sites and the target proteins include various protein kinases, receptor proteins, enzymes, structural and cytoskeletal proteins, and proteins involved in cell cycle control and apoptosis.13-15 14-3-3σ has been found frequently lost or decreased in human cancers of breast,16,17 liver,18 vulva,19 mouth,20 neuroendocrine,21 and prostate.22 This decrease was found to be due to hypermethylation of the GC rich region in the 14-3-3σ promoter.16,23-25 These findings are consistent with

Proteomic Analysis of NSCLC Cell Lines

research articles the spot B likely consists of M2. This finding is consistent with previous observation that M2 expression is increased in lung cancers.30 Because both spots A and C and part of the spot B represent the M1 isoform and all three spots have the same molecular mass but different pI, we speculate that the isoform M1 is likely modified posttranslationally. Furthermore, while the M2 isoform (spot B) is increased at protein level (Table 1) but not at the mRNA level (Figure 6B) in cancer cell lines compared to normal cells, it is possible that the expression of M2 is different at the translational level between normal and cancer cells.

Figure 6. Analysis of M-type pyruvate kinase expression by real time quantitative RT-PCR. Real time quantitative RT-PCR was performed as described in Experimental Procedures using primers specific to M1 (A) and M2 isoforms (B). Relative mRNA levels were measured using SYBR Green and calculated in the fold change (2∆Ct) relative to NHBE cells after normalization by the internal control, β-actin.

the results shown in this study and, together, suggest that 143-3σ may be a tumor suppressor of epithelial cells of multiple origins. However, in a previous study 14-3-3σ was found increased in lung cancers26 and in another study it was found deactivated in small cell lung cancer but not in NSCLC.25 Currently, the reason for these discrepancies of 14-3-3σ expression in lung cancers is not known. Although the mechanism how 14-3-3σ functions as a tumor suppressor is unknown, it has been shown that 14-3-3σ is strongly up-regulated upon exposure to ionizing radiation and DNA-damaging agent and appears to be essential for maintaining the G2/M checkpoint.27 Cells with decreased 14-3-3σ expression may develop mitotic catastrophe upon DNA damage. But the cells that do survive will have high probability to mutate and escape the growth restriction. The DNA damageinduced up-regulation of 14-3-3σ expression has been shown to occur through a p53 and BRCA1-mediated increase in transcription.28 It has also been shown that 14-3-3σ negatively regulate cell cycle progression by interacting with cyclindependent kinase 2.29 Thus, likely p53 and BRCA1 co-activates 14-3-3σ expression which in turn inhibits cyclin-dependent kinase 2 and leads to G2 arrest.2 There are two isoforms of M-type pyruvate kinases, M1 and M2, due to alternative mRNA splicing involving one of 11 exons which results in high identity in amino acid sequences of the two isoforms. The M2 isoform is known to be up-regulated in human lung30 and gastrointestinal cancers31 and is being developed as a diagnostic marker for lung cancers whereas no report on M1 and lung cancer is available. While two (A and C, Figure 5) of the three spots, all identified to be M-type pyruvate kinases, decreased in lung cancers, the third spot (B, Figure 5) increased at the protein level when compared with normal cells. On the basis of the peptide mass fingerprints, we found that while the spots A and C represent the M1 isoform,

Previously, it has been found that the pyruvate kinase M2 is phosphorylated at tyrosine residues in breast cancer patients.32 In rat tumor, the pyruvate kinase M2 was characterized to have the highest phosphate content (12 mol/mole protein) compared with the enzyme in normal liver (6 mol/mole protein).33 Although it is currently unknown whether the pyruvate kinase M1 is differentially phosphorylated in cancer, M1 was separated into three spots with different pI suggests that it may also be differentially phosphorylated in normal and cancer cells. It is also unknown whether the M1 isoform is involved in cancers. However, the finding that its expression is decreased in lung cancer cell lines suggests that it may potentially play some tumor suppressor role. We also found that intermediate filament proteins such as vimentin and cytokeratin 8 were up-regulated in the NSCLC cell lines. These proteins have been used as markers for lung cancer detection.34-38 Several other cytokeratins such as cytokeratin 5, 6, 14, 16, and 19 have also been found overexpressed in adenocarcinomas and squamous cell carcinomas of lung in the past.39 But, the overexpression of these proteins were not observed in this study. The elevated expression of both Y box-binding protein 1 (YB-1) and proliferating cell nuclear antigen (PCNA) in NSCLC cell lines are consistent with the previous observations that they were up-regulated in NSCLC.40 Human YB-1 is a member of a family of DNA binding proteins that contain a highly conserved domain which interacts with CCAAT boxes. Several human genes including PCNA contain a CCAAT box in their promoter region and, thus, can likely be activated by YB-1. YB-1 appears to play an essential role in cell proliferation and growth. Elevated expression of YB-1 has also been associated with poor prognosis of NSCLC patients. In this study, the protein spot which was identified to be YB-1 had an acidic pI (3.7), very different from the theoretical pI (9.9). However, the MALDITOF data showed a very high Z-score and very good peptide coverage of the matched protein. Because the observed molecular weight is larger than calculated, we propose that YB-1 protein may be modified extensively in NSCLC cells. Alternatively, it may also be due to different splicing of the YB-1 mRNA which may generate proteins of different size and pI. These possibilities await further investigation. The Nm23 gene was originally identified in melanoma cell lines with low metastatic potential and was thought to play a major role in suppressing tumor metastasis.41 In fact, the suppression of metastasis was demonstrated by transfecting the Nm23 gene into tumor cells with high metastatic potential.42 However, it was thought that Nm23 might not play any role in suppressing tumor metastasis in lung cancer.43 A positive correlation has been observed between the elevated level of nm23 expression and lung tumor stage.44,45 The elevated level of Nm23 was also found in the bronchial lavage fluid of patients Journal of Proteome Research • Vol. 3, No. 4, 2004 733

research articles with squamous cell lung cancer, suggesting that Nm23 may be developed as a diagnosis tool for lung cancer.46 hnRNP A2/B1 is a 31-kDa protein responsible for posttranscriptional regulation of gene expression by capping, splicing, polyadenylation, and cytoplasmic transport of mRNAs.47,48 The finding that hnRNP was overexpressed in lung cancer was first reported by Zhou et al.49 using a monoclonal antibody 703D4. In a clinical study using 103 bronchial lavage specimens, the use of 703D4 antibody to detect hnRNP A2/B1 was shown to be more accurate in detecting neoplasia than routine cytological examinations.50 On the basis of the finding that hnRNP may play an important role in lung development,51 it is tempting to propose that the up-regulated expression of hnRNP A2/B1 may be a potential cause of lung cancer. It is interesting to note that the elevated expression of hnRNP A2/ B1 has also been observed in human breast52 and pancreatic53 cancers. Heat shock protein 90β has been found up-regulated in some squamouse cell lung carcinomas,54 consistent with this study. HSP90β is also known to interact and stabilize several oncogenic protein kinases.55 However, whether the increased expression of HSP90β causes lung cancer is not known. In this study, only cell lines were used for proteomic analysis. Further study on whether the proteins (e.g., 14-3-3σ and TEF1δ) which are different between the cell lines are also altered in tumors compared with normal tissues would be necessary to determine their significance in lung tumorigenesis. Because tumor and normal tissue samples contain many cell types, the 2-DE data generated from tissues would be too complex for analysis. Furthermore, laser-assisted microdissection provides too little materials for 2-DE analysis. They, thus, are not well suited for the primary 2-DE analysis. However, these problems can be overcome by using cell lines which represent a pure population of cell types. It is currently unknown what causes the detected changes of the proteins observed in this study. However, these changes are presumably due to the difference in malignancy between the cells tested although it is not clear whether the changes are the cause or a result of the malignant transformation. It is also possible that the detected changes are due to the difference in growth cycle or rate between the normal and cancer cells. However, we think this possibility is unlikely because all cell lysate samples were prepared from cells grown at near confluency. It is still possible that the different growth conditions used to culture normal and cancer cells may cause the altered expression level of some of these proteins although most of these proteins (vimentin, cytokeratin 8, YB-1, PCNA, Nm23, hnRNP A2/B1, and HSP90β) are known lung cancer markers or known to be up-regulated in lung cancers which suggest that the differential expression may not be due to the differences in culture conditions. Clearly, further studies are needed to address these issues. In summary, we demonstrated in this study that the protein profiles between normal and NSCLC cell lines are different. Among 12 differentially expressed protein spots which were identified by MALDI-TOF and peptide mapping, 7 of them were known as markers of lung cancer or have been shown to be changed in lung cancers. Three of the 12 spots were identified to be M-type pyruvate kinase. Finally, the expression of the tumor suppressor gene 14-3-3σ is down regulated while that of the proto-oncogene TEF 1δ is up-regulated in NSCLC cells, which were confirmed by real time RT-PCR and western blot analyses. 734

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Abbreviations: 2-DE, two-dimensional electrophoresis; NSCLC, nonsmall cell lung cancer; SCLC, small cell lung cancer; SCC, squamous cell carcinoma; LCLC, large cell lung cancer; TEF1δ, translation elongation factor 1δ; YB-1, Y box-binding protein 1; PCNA, proliferating cell nuclear antigen; HSP, heat shock protein.

Acknowledgment. The authors wish to thank Dr. Mu Wang and his associates of the proteomics core at Indiana University School of Medicine for their technical assistance in PD Quest and MALDI-TOF analysis. This work was supported in part by National Institutes of Health grants CA64539 and CA94961. J.-T.Z. was a recipient of a Career Investigator Award from the American Lung Association. References (1) Srivastava, S.; Kramer, B. In Lung Cancer; Hansen, H. H., Ed.; Kluwer Academic Publishers: Norwell, 1994; pp 91-110. (2) Osada, H.; Takahashi, T. Oncogene 2002, 21, 7421-7434. (3) Merlo, A.; Herman, J. G.; Mao, L.; Lee, D. J.; Gabrielson, E.; Burger, P. C.; Baylin, S. B.; Sidransky, D. Nat. Med. 1995, 1, 686-692. (4) Hung, J.; Kishimoto, Y.; Sugio, K.; Virmani, A.; McIntire, D. D.; Minna, J. D.; Gazdar, A. F. JAMA 1995, 273, 558-563. (5) Bennett, W. P.; Colby, T. V.; Travis, W. D.; Borkowski, A.; Jones, R. T.; Lane, D. P.; Metcalf, R. A.; Samet, J. M.; Takeshima, Y.; Gu, J. R.; et al. Cancer Res. 1993, 53, 4817-4822. (6) Kishimoto, Y.; Sugio, K.; Hung, J. Y.; Virmani, A. K.; McIntire, D. D.; Minna, J. D.; Gazdar, A. F. J. Natl. Cancer Inst. 1995, 87, 12241229. (7) Sozzi, G.; Miozzo, M.; Donghi, R.; Pilotti, S.; Cariani, C. T.; Pastorino, U.; Della Porta, G.; Pierotti, M. A. Cancer Res. 1992, 52, 6079-6082. (8) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (9) Decker, E. D.; Zhang, Y.; Cocklin, R. R.; Witzmann, F. A.; Wang, M. Proteomics 2003, 3, 2019-2027. (10) Pincheira, R.; Chen, Q.; Zhang, J. T. Br. J. Cancer 2001, 84, 15201527. (11) Joseph, P.; Lei, Y. X.; Whong, W. Z.; Ong, T. M. J. Biol. Chem. 2002, 277, 6131-6136. (12) Prasad, G. L.; Valverius, E. M.; McDuffie, E.; Cooper, H. L. Cell Growth Differ. 1992, 3, 507-513. (13) van Hemert, M. J.; Steensma, H. Y.; van Heusden, G. P. BioEssays 2001, 23, 936-946. (14) Rosenquist, M. Braz. J. Med. Biol. Res. 2003, 36, 403-408. (15) Fu, H.; Subramanian, R. R.; Masters, S. C. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 617-647. (16) Ferguson, A. T.; Evron, E.; Umbricht, C. B.; Pandita, T. K.; Chan, T. A.; Hermeking, H.; Marks, J. R.; Lambers, A. R.; Futreal, P. A.; Stampfer, M. R.; Sukumar, S. PNAS 2000, 97, 6049-6054. (17) Vercoutter-Edouart, A.-S.; Lemoine, J.; Le Bourhis, X.; Louis, H.; Boilly, B.; Nurcombe, V.; Revillion, F.; Peyrat, J.-P.; Hondermarck, H. Cancer Res. 2001, 61, 76-80. (18) Iwata, N.; Yamamoto, H.; Sasaki, S.; Itoh, F.; Suzuki, H.; Kikuchi, T.; Kaneto, H.; Iku, S.; Ozeki, I.; Karino, Y.; Satoh, T.; Toyota, J.; Satoh, M.; Endo, T.; Imai, K. Oncogene 2000, 19, 5298-5302. (19) Gasco, M.; Sullivan, A.; Repellin, C.; Brooks, L.; Farrell, P. J.; Tidy, J. A.; Dunne, B.; Gusterson, B.; Evans, D. J.; Crook, T. Oncogene 2002, 21, 1876-1881. (20) Gasco, M.; Bell, A. K.; Heath, V.; Sullivan, A.; Smith, P.; Hiller, L.; Yulug, I.; Numico, G.; Merlano, M.; Farrell, P. J.; Tavassoli, M.; Gusterson, B.; Crook, T. Cancer Res. 2002, 62, 2072-2076. (21) Yatabe, Y.; Osada, H.; Tatematsu, Y.; Mitsudomi, T.; Takahashi, T. Oncogene 2002, 21, 8310-8319. (22) Cheng, L.; Pan, C. X.; Zhang, J. T.; Zhang, S.; Kinch, M. S.; Li, L.; Lee, A. B.; Wade, C.; Koch, M. O.; Ulbright, T. M.; Eble, J. N. Clinical Cancer Res. 2004, in press. (23) Suzuki, H.; Itoh, F.; Toyota, M.; Kikuchi, T.; Kakiuchi, H.; Imai, K. Cancer Res. 2000, 60, 4353-4357. (24) Umbricht, C. B.; Evron, E.; Gabrielson, E.; Ferguson, A.; Marks, J.; Sukumar, S. Oncogene 2001, 20, 3348-3353. (25) Osada, H.; Tatematsu, Y.; Yatabe, Y.; Nakagawa, T.; Konishi, H.; Harano, T.; Tezel, E.; Takada, M.; Takahashi, T. Oncogene 2002, 21, 2418-2424. (26) Nakanishi, K.; Hashizume, S.; Kato, M.; Honjoh, T.; Setoguchi, Y.; Yasumoto, K. Hum. Antibodies 1997, 8, 189-194.

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