| 1. BACKGROUNDLinzhou City (former Linxian) and its nearby counties, Huixian and Anyang in Henan Province, have the highest incidence and mortality rates for esophageal cancer (EC) in China as well as in the world. EC remains the leading cause of cancer-related death in these areas. Although studies have shown the changes of some EC-associated molecules, the key molecular mechanisms in esophageal carcinogenesis are largely unknown. Furthermore, high specific and sensitive biomarkers for high-risk subject screening and early detection of EC haven't been identified. Recent studies suggest that, during multi stage carcarcinogenesis, epithelial cells could abnormally express some proteins that often exist only in embryo development other than in the well differentiated tissues, which could be clinically used as molecular markers for early tumor diagnosis, such as AFP in liover cancer, etc. The genesis of fetal esophageal epithelia has been proposed to develop from nascent simple columnar epithelia cells to stratified squamous epithelium in which the well differentiation is gradually formed. In this process, the proliferous nascent epithelia cells tend to be differentiated and partitioned to basal stem cells, middle proliferous cells, reserve cells, epithelia cells and desquamative cells. This genesic process is quite different from that of epithelia carcinogenesis in which the well differentiated epithelia cells extraordinarily undergo hyperproliferation, loss of differentiation and finally malignant transformation. The comparative study of fetal and adult esophagus epithelia on the morphology, the molecular changes and their relationships with cell proliferation and differentiation will contribute to the illumination of the exact mechanisms of epithelial carcinogenesis, the identification of molecular biomarkers for early diagnosis and high-risk subject screening. However, the comparative studies on the molecular changes in multiple stages of fetal and adult esophageal epithelia in the processes of cancerization are still not available. Our previous research and other reports have revealed that the abnormality of tumor suppressor gene p53-Rb has the highest frequency in early esophageal cancer and precancerous lesions in high-risk areas of Henan. In addition, the serum protein analysis also indicates the remarkble protein level variations in EC and precancerous lesions. Based on the these progresses, the present studies were thus undertaken to characterize the molecular and morphological changes related with cell proliferation in mature differentiation of fetal esopgageal epithelia with defferent ages from 3-10 months and in loss of defferentiation of adult esophageal multistage carcinogenesis with proteomic analysis, Dot blot, RT-PCR, immunohistochemistry, electron microscopy and histopathology. The specific aim for this study was to identify molecular biomarkers related with abnormal differentiation and cell proliferation for high-risk subject screening and early detection and to facilitate the understanding of the mechanisms of human esophageal carcinogenesis.2. MATERIALS and METHODS2.1 Subjects2.1.1 Fetal esophagus The 172 cases of fetal esophageal epithelial samples (male 61, female 109,2 Nonsexsuality) were obtained from Huixian Family Planing Center, including 31 cases (18%) at 3 months, 54(31%) at 4 months, 29 (17%) at 5 months, 24 (14%) at 6 months, 15 (8%) at 7 months, 13 (8%) at 8 months, 4 (2%) at 9 months, and 1 (1%) at 10 months.2.1.2 Adult esophagusThe 17 cases (10 males and 7 females with a mean age of 60±7) of adult esophageal epithelia samples were obtained from Linzhou Central Hospital of Henan Province and Yaocun Esophageal Tumor Hospital. All the patients were not received any chemotherapy or radiotherapy before surgery.2.2 Sample collection and processing2.2.1 Fetal esophagusThe fetal esophagus specimens were obtained from legal abortion or induced abortion with prostaglandinum. The gestational ages were determined by the last catamenia time before pregnancy, and verified by measuring the full length of the embryo and observation of the morphology of fetal hands and feet. The whole esophagus and one third of upper stomach were collected within 2 hrs after abortion or induced abortion, and splitted vertically. Half sample was fixed with 85% ethanol. The rest half was frozen in nitrogen and then stored at -80℃for extraction of protein, DNA and RNA.2.2.2 Adult esophagusThe surgically resected esophageal cancer specimen was splitted vertically within 2 hrs. Based on the theory that the actively hyperplastic esophageal epithelium was lightly stained or negative for Lugol solution staining, half of the specimen was stained with Lugol solution and sampled from stained and unstained areas 5 cm away, or from the cutting edge. The following treatments for both half of the specimen were the same as fetal esophagus.2.2.3 Electron microscopySpecimens of 1mm3 size were taken from upper, middle and bottom segments of the fetal esophageal mucosa and fixed with 2.5% neutral glutaraldehyde. For transmission electron microscopy, the samples were further fixed with osmium acid, and then dehydrated with gradient acetone, embedded in Epon812 rosin, and finally stained with uranyl acetate and lead citrate. For scan electron microscopy, samples were fixed with 2.5% neutral glutaraldehyde and further fixed with 1% osmium acid. After dehydrated with gradient ethanol, samples were dried and gilded.2.3 Criteria for histopathological diagnosisFor fetal esophagus, the thickness of epithelium, layers of cells and apoptotic cell fraction were examined under microscopy after HE staining.For adult esophagus, with the diagnosis criteria established previously by us based on cellular morphology, tissue structure and cell differentiation, epithelia were classified into normal (NOR), basal cell hyperplasia (BCH), dysplasia (DYS), carcinoma in situ (CIS), well differentiated squamous cell carcinoma (SCC), moderately differentiated SCC and poorly differentiated SCC.2.4 Morphometrical analysis on fetal esophageal epithelia2.4.1 Optical microscope image analysisMicroscopical sizing apparatus was used to measure the fetal esophageal epithelium after fixed with ethanol and stained with HE. All the data were statistically analyzed.2.4.2 Electron microscopyElectron microscopy was performed with transmission electron microscope (JEM1230, Japan Electron Co.) and scan electron microscope (AMRAY-1000B, Engineering Resources Co. USA).2.5 ImmunohistochemistryImmunohistochemistry was performed with Avidin Biotin Complex (ABC) method.2.6 Extraction of RNA and RT-PCRotal RNA was extracted with TRIzol (GibocoBRL, Life technologies, USA). The ratio of A260/A280 was measured for the evaluation of integrity and purity, which was ranging between 1.6 and 1.8 for all the RNA samples in this experiment. RNA was reverse transcribed with SuperScriptTM First-Strand Synthesis System (Invitrogen, USA). The reaction volume of 25μl contained 3μg RNA, 0.5 mM dNTPs, 0.025μg/ul oligo dT, 5μM MgC12, 0.01 M DTT, 2 U/μL RNaseOUT TM and 2.SU/μL SSâ…¡RT. For following PCR, the 30μl of reaction volume containing 1.5 mM MgCl2, 0.1 mM dNTPs, 0.05 U Taq DNA polymerase and primers (DNP,p21,p14,p15,RASSF1) was pre-denatured at 94℃for 10 min, followed by 35 cycles at 94℃for 30s, 55℃for 1min and 72℃for lmin, finally elongated at 72℃for 10min.2.7 Methods for proteomics2.7.1 Protein extraction and measurement of concentrationTissue was treated with lysis buffer (7% mol/L of urea, 2 mol/L of Thiourea, 4% Chaps, 65 mmol/L of DTT, 0.1 g/L of PMSF, 0.001g/L of Aprotinin, 0.05 g/L of Rnase and 0.2 g/L of Dnase)(1 mg of tissue adding 2μl lysis buffer) then homogenized for 5 to 10 rain. Tissue extracts were sonicated on ice at 400 W for ls, 100 time repeats with 2s of intervals, and centrifuged at 15000g for 15 min at 4℃to get rid of the precipitates. After further centrifuged at 15000g for 15 rain at 4℃, supematant was transferred to a new Eppendorf tube, allocated and stored at -80℃for further analysis. Total proteins were quantified by the Bradford method according to the protocol.2.7.2 Two-D-electrophoresis, image analysis, MALDI-TOF analysis and protein identificationIEF was conducted using Amersham Biosciences IPGphor. Second dimensional separation was performed by SDS-PAGE and stained with silver nitrate. Images of 2-D gels were digitalized with ImageScancer GS-800 (Bio-Brad). Image analyses were conducted with ImageMaster 2-D Elite software (PD-Quest 7.1, Bio-Brad). The normalized value for each protein spot volume was used for comparison. Spots of interest were cut out with a clean scalpel and subjected to in-gel digestion with trypsin. A MALDI-TOF mass spectrometer were used for the specific peptide mass fingerprint (PMF). Proteins were identified by PMF and searching the NCBI protein database.2.8 ImmmunoblottingAfter identification of candidate proteins following peptide fingerprinting and the database, the protein of interest was selected for Western blotting to confirm the results of protein database searching. After 1- or 2-DE, Proteins were then transferred to PVDF membranes by semi-dry transfer apparatus at 0.8 mA/cm2 for 1 h. The membranes were washed with PBS-Tween and blocked with 5% fat-free milk in PBS-Tween for overnight at at 4℃and incubated with an antibody of DNP (1:300 dilution in a 5% fat-free milk solution) at 4℃overnight. After washing, the membranes were finally visualized by DAB methods.2.9 Statistical analysisData was analyzed with SPSS10.0 softeware for x2 and Kappa tests. For ImageMaster 2-D Elite analysis, the protein spot volume and its variation, the one-tailed Student's t test was used to analyse the data. P＜0.05 was considered statistically significant.3.RESULTS3.1 Histopathological observation:Fetal esophagus: The esophageal epithelium at 3 months has been observed with stratified squamous epithelium with 2-5 layers of cells with similar cell morphology, which were similar as the pooly differentiated SCC cells. For 4 to 6-month, the esophageal cell layers were up to 5-8; the basement lamina cells in some parts were up to 3 layers with out-of-order arrangement and exhibited colunmar and cuboid morphology. The cytoplasm of these cells was strongly basophilic, and the karyon was big and dark granular colored. Apoptotic cells were also scattered in these areas. For the esophageal epithelium with 5 to 10 months, the layers of epithelia and basal lamina cells were 6 to 10 and 1 to 2, respectively, in which cells were well arranged and apoptotic cells were still found sporadically. One cases with 6 month gestational age showed the squamous epithelim intermingled with columnar epithelium like adult Barrett's esophagus. Esophageal gland ducts could be found in epithelium with 7-month fetus.Adult esophagus: Based on lugol staining, two pieces of tissue (one from cancer, another from the adjacent to cancer) were obtained from each SCC specimen (n=17). Based on precancerous lesions detected, these samples were divided into two groups. Group 1: including 15 cases with SCC and corresponding normal esophageal mucosa, and SCC was sub-grouped to well, moderately and poorly differentiated SCC; Group 2: including 2 cases with SCC and the corresponding precancerous lesions (BCH, DYS and CIS). Case 23 with SCC had 1 DYS and 1 CIS. Case 29 with SCC had 1 BCH and 1 DYS.3.2 Uultrastructure of fetal epithefiumFetal epithelial cells had abundant dissociated ribosomes and hepatin, but lacked of organelles in cytoplasm. Tonofilaments gradually increased in the cells from basal to superficial epithelial cells. The gap junctions also increased along with the increasing of gestational ages in the cells from basal to superficial cells, which was quite different with the desmosome junction.3.3 Immunohistochemical analysisThe positive cell numbers for PCNA, pl6, CK13 and Ck7 were very low in epithelium with 3-month gestational age and increased with fetal aging. The positive rates and the positive cell numbers were significantly different between 3-month and 5, 6, 7, 8, 9-month. Interestingly, p16, CK13 and CK7 proteins were positively expressed in adult normal esophageal paracancer tissue, and the positive cell number gradually decreased from BCH to DYS, CIS, well differentiated SCC, moderately differentiated SCC and poorly differentiated SCC, while the PCNA protein was poorly expressed in adult normal esophageal paracancerous tissue, and the positive cell number gradually increased from BCH to DYS and CIS, and from well differentiated to moderately differentiated and poorly differentiated SCC. P16, CK13 and CK7 protein expression had the opposite varying tendency in the process of fetal esophageal epithelial development and in the carcinogenesis of esophagus. However, the changing pattern was the same for PCNA. The immunostaining for RASSF1, p21, p14 and p15 was negative in all the esophageal epithelium with various gestational ages.3.4 RT-PCRHemi-quantitative PCNA RT-PCR for fetal esophageal epithelium showed that mRNA increased along with gestational age increasing. These results correlated well with that in normal tissue, precancerous lessions and cancer. The p16 mRNA was high in fetal esophageal epithelium, but low in normal tissue, precancerous lessions and cancer. The p15, p14 and p21 mRNAs were expressed significantly in normal tissue, precancerous lessions and cancer, but negatively expressed in fetal esophageal epithelium with various gestational ages.3.5 Proteomic analysisThe 2-DE proteomic analysis indicated that there were 26 protein spots with significantly expression and these proteins were subjected to further MALDI-TOF analysis. After PMF and searching the NCBInr protein database, 21 proteins were identified. Some of these proteins had isomers such as triosephosphate isomerseâ… , thyroid hormone receptor interactor 11 and isoform CRAb, etc. Out of the 21 proteins, 11 were over expressed in cancer and fetal esophageal epithelium. However, these proteins decreased or absent in normal epithelium such as crystal structure of recombinant glutathione transferase Chain A, Dermatopontin(DPN), Heat shock protein 27, calcium-binding protein S100A9, immunoglobulin heavy chain variable region, HCG1997574, phosphoglycerate mutase 1, peroxiredoxin 6, human triosephosphate isomerase of new crystal form chain A, actin, alpha, cardiac muscle isoform, disabled homolog 1 and so on.The DPN, S100 calcium-binding protein A9 and heat shock protein 27 proteins which were negative in adult normal epithelium were simultaneously expressed in fetal esophageal epithelium and cancer, suggesting that these proteins may be related to with epithelial hyperproliferation.3.6 Western blot analysisWith western blotting, DPN protein was identified based on proteomics, and it revealed that the over expression for DPN existed in esophageal cancer and precancerous lessions other than the normal epithelium.4. CONCLUSINS4.1 This study have showed that there are active proliferated cells and apoptotic cells existed in fetal esophageal epithelia. The squamous epithelium is intermingled with columnar epithelium similar as in adult Barrett's esophagus. Apoptotic ceils also appeare in esophageal cancer and precancerous lessions. All these results suggest a similar morphology changes in adult esophageal multistage carcinogenesis and fetal esophageal epithelial development. The comparative study on ultrastructure of fetal esophageal epithelium and of the esophageal cancer, precancerous lessions and normal epithelium demonstrates that the sub-cellular structures and the cell junctions in fetal esophageal epithelium are identical with that in esophageal cancer and precancerous lessions.4.2 The over expressed PCNA, p16, CK7 and CK13 proteins consistent with the gestational ages suggest that p16, CK7 and CK13 proteins could be poorly expressed in esophageal cancer and precancerous lessions. PCNA expression is identical in both tissues. PCNA, p16, CK7 and CK13 proteins play important roles in fetal esophageal development and adult esophageal carcinogenesis. The absence of P14, p15 and p21 protein in fetal esophageal epithelium indicate that they may not contribute to the epithelia differentiation. However, in esophageal cancer and precancerous lessions, these proteins are over expressed, suggesting that the molecular mechanisms of fetal esopgageal epithelium differentiation are not completely identical with that of loss of differentiation though certain similarities existed in some of the stages.4.3 The comparative studies on adult normal epithelium, esophageal cancer and precancerous lesions, fetal esophageal epithelia with various gestational ages with 2-DE based proteomics, MALDI-TOF analysis and Bioinformatics demonstrate 21 proteins associated with carcinogenesis. These proteins are also related with cell and tissue structure, energy metabolisms, protein synthesis, cell differentiation, apoptosis, cell adhesion and tumor metastasis.4.4 The proteins associated with esophageal carcinogenesis identified in this study can not only improved the understanding of molecular mechanisms that esophageal carcinogenesis involves in multiple factors, multiple genes and multiple stages, furthermore, they may provide more candidates of biomarkers for early detection, early diagnosis of esophageal cancer and the selection of new therapeutic targets.4.5 The changes of DPN protein in fetal esophageal epithelium suggeste that this kind of protein could have profound meanings in precancerous lesions. Its high and low levels of expression may be related to the progression and reversal of precancerous lesions and also may provide molecular markers for early screening and diagnosis of esophageal cancer in high-risk populations.
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