Identification of differentially expressed proteins between human esophageal immortalized and carcinomatous cell lines by two-dimensional electrophoresis and MALDI-TOF-mass spectrometry

Xing-Dong Xiong , Li-Yan Xu , Zhong-Ying Shen , Wei-Jia Cai, Jian-Min Luo, Ya-Li Han,  En-Min Li

Xing-Dong Xiong , Jian-Min Luo, En-Min Li, Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515031, Guangdong Province, China
Li-Yan Xu , Zhong-Ying Shen , Wei-Jia Cai, Department of Pathology, Shantou University Medical College, Shantou 515031, Guangdong Province, China
Ya-Li Han, Department of Biology, Shantou University, Shantou 515031, Guangdong Province, China
Supported by National Natural Science Foundation of China, NO.39900069, NO.30170428; Guangdong Provincial Natural Science Foundation, NO.990799, NO.010431; Guangdong provincial College Natural Science Foundation, NO.200033; Guangdong provincial medical Scientific Foundation, NO.A2001419 and Research and Development Foundation of Shantou University, NO.L0004, NO.L00012.
Corresponding to: Dr. En-Min Li, Department of Biochemistry and Molecular Biology, Shantou University Medical College, 22 Xinling Road, Shantou 515031, Guangdong Province, China.  nmli@21cn.com
Telephone: +86-754-8532720
Received  2002-05-18    Accepted  2002-06-27


Abstract
AIM: To identify the differentially expressed proteins between the human immortalized esophageal epithelial cell line (SHEE) and the malignant transformed esophageal carcinoma cell line (SHEEC), and to explore new ways for studying esophageal carcinoma associated genes.

METHODS: SHEE and SHEEC cell lines were used to separate differentially expressed proteins by two-dimensional electrophoresis. The silver-stained 2-D gels was scanned with EDAS290 digital camera system and analyzed with the PDQuest 6.2 Software. Six spots in which the differentially expressed protein was more obvious were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).

RESULTS: There were 1074.58 and 1159.91 protein spots observed in SHEE and SHEEC respectively, and the majority of these spots between the two cell lines matched each other (r=0.772), only a few were expressed differentially. After analyzed by MALDI-TOF-MS and database search for the six differentially expressed proteins, One new protein as well as other five sequence-known proteins including RNPEP-like protein, human rRNA gene upstream sequence binding transcription factor, uracil DNA glycosylase, Annexin A2 and p300/CBP-associated factor were preliminarily identified.

CONCLUSION: These differentially expressed proteins might play an importance role during malignant transformation of SHEEC from SHEE. The identification of these proteins may serve as a new way for studying esophageal carcinoma associated genes.

Xiong XD, Xu LY, Shen ZY, Cai WJ, Luo JM, Han YL, Li EM. Identification of differentially expressed proteins between human esophageal immortalized and carcinomatous cell lines by two-dimensional electrophoresis and MALDI-TOF-mass spectrometry. World J Gastroenterol  2002; 8(5): 777-781



INTRODUCTION
Since Wilkins and Williams first proposed the concept of "roteome" in 1994, the studies on tumor proteome have been made mighty advances^[1]. It is expected to find new special tumor markers and clone their associated genes via separating and identifying the tumor differentially expressed proteins by the proteomic approach to reveal the tumor pathogenesis and carry out the gene
therapy^[2-6].
      Esophageal carcinoma is one of the most common malignant tumors in China^[7-19], and its etiology and pathogenesis remain to be determined^[20-23]. Recent studies are mainly focused on the relationship between the change of oncogenes/suppressor oncogenes and esophageal carcinoma. However, there is no strong evidence to indicate that these oncogenes and suppressor oncogenes, including myc, ras, EGFR, int-2, cyclin D1, p53, Rb, p16, MCC, APC which are cloned originally from other kinds of tumors, are closely related to the esophageal carcinoma^[24-28]. Therefore, it is necessary to clone the new oncogenes or suppressor oncogenes, which might have an more intimate relationship with esophageal tumor pathogenesis, directly from esophageal carcinoma tissues or cells.
      In recent years, it has been increasingly concerned about the roles of the human papilloma virus (HPV) played in the esophageal carcinogenesis^[29-32]. In our previous work, we transfected human embryonic esophageal mucosa cells with HPV18 E6E7 genes, and established an immortalized epithelial cell line SHEE^[33,34]. The SHEE cells were further exposed to the tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate,TPA) to be induced malignant transformation, and from which a human embryonic esophageal epithelial carcinoma cell line SHEEC was then established^[35,36]. These studies not only provided the evidence for the close relationship between HPV and the esophageal carcinogenesis, but also established a reliable model for studying the molecular mechanisms of esophageal carcinogenesis, and cloning new esophageal carcinoma associated genes. In the present study, the differential expression of proteins between SHEE and SHEEC was investigated by the proteomic approach including two-dimensional electrophoresis and MALDI-TOF-MS, which might serve as a new way for studying esophageal carcinoma associated genes.

MATERIALS AND METHODS
Cell culture
SHEE and SHEEC were cultured in MEM medium (Gibco) supplemented with 100 ml/L fetal borine serum (100 u/ml penicillin, 100 u/ml streptomycin) and incubated at 37 ℃ in humidified atmosphere of 50 ml/L CO₂ incubator.

Whole soluble protein extraction and pre-treatment
To obtain whole soluble protein, the experimental procedures in Molecular cloning (2nd ed.) were employed^[37]. Briefly, when the cultured cells grew into a full monolayer, they were washed with ice-cold phosphate-buffer saline (PBS) three times and then treated with cold buffer containing 50 mmol/L Tris-HCl, pH8.0, 150 mmol/L NaCl, 1 %Triton X-100, 100 mg/ml Phenylmethylsulfonyl fluoride (PMSF) for 20 min at 4 ℃. The broken cells were collected with a scraper and centrifuged at 12 000 g for 5 min. The supernatant, which contained the whole soluble proteins, was added to Micro Bio-Spin chromatography columns, and the purified sample was obtained after centrifugation at 1 000g for 4 min. Protein concentrations were determined by Bradford method (BIOPhotometer, Eppendorf). The sample aliquots were stored at -20 ℃ until used.

Two-dimensional electrophoresis
Two-dimensional electrophoresis was carried out by using the Mini-PROTEAN II 2-D apparatus (Bio-Rad). 70 mg of the whole soluble proteins were mixed with the rehydration solution containing 8 mol/LUrea, 4 %CHAPS,10 mmol/L DTT,0.2 %(w/v) IPG buffer (pH3-10,liner) to a total volume of 125 ml. The mixture was pipetted into IPG strip tray channels. Both the rehydration and focusing were performed in the same focusing tray. IPG dry strips (pH3-10, 7 cm) were lowered onto the mixture with the gel side down, and then covered with mineral oil. The rehydration and isoelectric focusing (IEF) were done as follows: 1) rehydration for 12-14h, 0V; 2) 250V, 30 min; 3) 250V to 4 000V, 2 h; 4) 4 000V, 5 h. All the procedures above were performed at 20 ℃. After IEF separation, the strips were immediately equilibrated for 210 min with 6 mol/L Urea, 0.375 mol/L Tris-HCl (pH8.8), 2 % SDS and 20 % glycerol. In the first equilibration solution, 2 %(w/v) DTT was included, and 2.5 %(w/v) iodoacetamide was added in the second equilibration. Then the IPG strips were placed on a 1.0 mm thick, 12 % SDS-PAGE gel and sealed with 1 % LowMelt agarose. Electrophoresis was carried out at a constant voltage (40 mV/gel) until the bromophenol blue frontier reached the bottom of the gels about 0.5 cm. After electrophoresis, the SDS-PAGE gels were stained with silver stain plus kit (Bio-Bad).

Image acquisition and analysis
Image scanning for the silver-stained 2-D gels was performed with EDAS290 digital camera system (Kodak) and image analysis with the PDQuest 6.2 Software (Bio-Rad). To get reliable results, three gels were employed for each cell line. After the background subtraction, spot detection and match, one standard gel for each cell line was obtained. These standard gels were then matched to yield information about the spots of differentially expressed proteins.

Protein identification by MALDI-TOF-MS
Six spots in which the differentially expressed protein was more obvious in each cell line were cut out from the gel. After washed with 300 ml milliQ water for 15 min, each protein spot was decolorized with the successive action of 50 ml of 15 mmol/L Potassium ferricyanide and 50 mmol/L sodium thiosulphate for 5-10 min. The faded gel pieces were dried in a vacuum centrifuge tube for 5 min. The cysteine reduction and alkylation were performed as incubated with 10 mmol/L DTT, 100 mmol/LNH4HCO₃  at 56 ℃ for 1 h in the dark. The gel pieces were then dried again and incubated with 50 mmol/L fresh iodoacetamide in 100 mmol/L NH4HCO₃ at room temperature for 30 min. Thereafter the gel pieces were rehydrated in digestion buffer containing 20 ml of 12.5 mg/ml modified trypsin and 20 mmol/L NH4HCO₃ for 30 min in ice. The excess liquid was removed and the gel pieces were digested continuously at 30 ℃ overnight(>16 h). The resulting peptide mixture was extracted from the digested solution by centrifugation and then resuspended in 10 ml of 50 % CH3CN and 0.1 % trifluoroacetic acid (TFA) for 10min at 30 ℃ on a shaking platform. Peptide mass maps were generated by Applied Biosystems Voyager System 6192 MALDI-TOF-mass spectrometry (ABI,USA). Peptide masses were analyzed using the MS-Fit search program (http://prospector.ucsf.edu/ucsfhtml4.0u/msfit.htm).

RESULTS
2-D map and image analysis
The whole soluble proteins of SHEE and SHEEC were extracted in one step and desalted with Micro Bio-Spin chromatography columns, which made the 2-D electrophoretic patterns a much higher quality (to be published). Three pairs of gels from different batches of SHEE and SHEEC were analyzed for the purpose of quantitative and qualitative comparison with the software PDQuest6.2. There were 107 4.58 and 1159.91 protein spots observed in SHEE and SHEEC respectively, and the majority of these spots between the two cell lines matched each other (r=0.772), only a few were expressed differentially. Six spots in which the differentially expressed protein was more obvious were selected and analyzed with MALDI-TOF-MS. The spot 1 was only expressed in the samples of SHEEC and absent in that of SHEE. In contrast, the spots 2 to 6 were merely observed in the SHEE samples. These six spots were marked with arrows at the corresponding site in Figure 1.

Figure 1  The differentially expressed protein spots observed in SHEE (left) and SHEEC (right) two-dimensional gels samples

MALDI-TOF-MS analysis and protein identification
The proteins contained in the six spots were identified respectively by MALDI-TOF-MS on the basis of peptide mass matching; In this way, the peptide mass fingerprinting map for each protein spot was obtained as show in Figure 2. The experimental data revealed that the protein in spot 6 was with an unknown sequence (Figure 3), and its characteristics remains to be investigated. The identified protein names, accession numbers, as well as the number of the matching peptide, the theoretical Mr and pI values, i.e. for each protein spot were listed in Table 1.

Table 1  Protein identified by mass spectrometry

Mode of operation: Reflector    Extraction mode: delayed            Accelerating voltage: 20000 V
Acquisition mass range: 750-3500Da   Number of laser shots: 150/spectrum   Laser intensity: 2233
Figure 2  The MALDI-TOF mass spectrum map of protein spot 1

Mode of operation: Reflector          Extraction mode: delayed            Accelerating voltage: 20000 V
Acquisition mass range: 750-3000Da    Number of laser shots: 200/spectrum   Laser intensity: 2224
Figure 3  The MALDI-TOF mass spectrum map of protein spot 6


DISCUSSION
The investigation of differentially expressed proteins that occured during the generation and development of tumors is a new effective way to study tumor associated genes^[38,39]. In the present study, we preliminarily studied several of the differentially expressed proteins between SHEE and SHEEC with two-dimensional electrophoresis and MALDI-TOF-mass spectrometry. By comparing the reference proteins or peptides, five sequence-known proteins and a novel sequence-unknown protein, which expressed more differentially in the course of malignant transformation of esophageal epithelial cells, were identified for the first time. It is therefore further indicated that the methods adopted in the present study could provide a new way to study esophageal carcinoma associated genes.
      RNPEP-like protein being composed of 494 amino acid residues has 49 % identity with aminopeptidase B in amino acid sequence^[40]. As judged by the parameters such as the numbers and intensity of peptide matching peak, the sequence coverage of matching peptide, as well as the theoretical and approximate values of  Mr and pI, RNPEP-like protein was considered as the much higher expression in SHEEC cells. However, the significance of its overexpression in SHEEC remains unknown.
     Human rRNA gene upstream sequence binding transcription factor (hUBF) is a critical element in the regulation of rRNA transcription, which performs its function by binding to the rRNA gene upstream regulator sequence (-200 to -107 and -45 to 20)^[41]. In our present work, it has been shown that the down-regulation of hUBF expression was obvious in the course of malignant transformation of SHEE, which might be the results of alterations in the regulation mechanism of rRNA gene transcription occurred in the malignant transformation of the human immortalized esophageal epithelial cells. In addition, whether or not the fact that diminished hUBF expression has been found in the well-differentiated teratocarcinoma cells^[42] related hUBF to the neoplastic differentiation, remain to be investigated.
      Uracil DNA glycosylase (UDG) is an enzyme for the DNA repairment. It can hydrolyse the N-glycosidic bond connecting the base to the deoxyribose and release free uracil base and DNA with an abasic site as its products^[43]. Moon and his associates^[44] found that the uracil DNA glycosylase gene (UNG) in sporadic glioblastomas had a point mutation in exon 3, and concluded that the genetic alterations of UNG might play an important role in the development of primary glioblastomas. In our study, the UDG expression shows absent or too low to be detected after SHEE malignantly transformed into SHEEC, which means that there might exist repairing deficiencies for the damaged DNA in the course of malignant transformation of the human immortalized esophageal epithelial cells.
      Annexin A2 belongs to the family of annexins that bind to phospholipids in a calcium-dependent manner. So far at least 13 annexin family members have been found. There are  four repeats of a 70 amino acid motif and a variable N-terminal end contained in all these annexin family members. According to lots of investigations, the annexins seem to be involved in various biological processes including endocytosis, exocytosis, the phospholipase A2 inhibition as well as  ion channel and protein kinase C activity^[45,46]. In recent years, it has been found that the annexin I was overexpressed in some kinds of malignant tumors such as human hepatocellular^[47] and pulmonary carcinomas^[48], which suggests that some of the annexin family members might relate to the carcinogenesis. Moreover, Chetcuti and his associates^[49] found that the annexin II was expressed in the nomal and benign hyperplastic prostate tissue, and absent in all prostate cancer specimens. These results indicated that different members of the annexin family may have varied roles in the development and progression of tumors. The fact that the annexin A2 was expressed in SHEE but absent in SHEEC in our research indicates that annexin A2 might play a role of suppressor oncogene during malignant transformation of the human immortalized esophageal epithelial cells.
      The p300/CBP-associated factor (P/CAF) that possessed intrinsic histone acetylase activity could regulate the gene expression of various sequence-specific factors that are involved in cell growth and/or differentiation including CREB, c-Jun, Fos and c-Myb through promoter-specific histone acetylation. Yang et al^[50] found that the expression of P/CAF in HeLa cells could block the cell-cycle progression from G1 to S phase, and counteract the mitogenic activity of adenoviral oncoprotein E1A. The P/CAF expression was absent in the esophageal carcinoma cells found in our study suggests that the P/CAF might play a role in the suppressing of the esophageal carcinoma development.

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Edited by Zhu L