Comparative Proteome Analysis of Human Lung Squamous Carcinoma Tissue

Objective： To establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression tumor-associated proteins by using proteome analysis. Methods： Comparative proteome analysis with 20 human lung squamous carcinoma tissues and the paired normal bronchial epithelial tissues adjacent to tumors was carried out. The total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis （2-DE） and silver staining. The differential expression proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Results： （1） Well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained. For tumor tissue, average spots of 3 gels were 1567±46, and 1436±54 spots were matched with an average matching rate of 91.6%. For control, average spots of 3 gels were 1349±58, and 1228±35 spots were matched with an average matching rate of 91.03%. The average position deviation of matched spots was 0.924±0.128 mm in IEF direction, and 1.022±0.205 mm in SDS-PAGE direction; （2） A total of 1178±56 spots were matched between the eleetrophoretie maps of 20 human lung squamous carcinoma tissues and paired normal tumor-adjacent bronchial epithelial tissues. Seventy-six differentially expressed proteins were screened; （3） Sixty-eight differential proteins were identified by PMF, some proteins were the products of oneogenes, and others involved in the regulation of cell cycle and signal transduetion; （4） In order to validate the reliability of the identified results, the expression of 3 proteins mdm2, c-jun and EGFR, which was correlated with lung squamous carcinoma, was detected by immunohistoehemieal staining and Western blot analysis. The results revealed that mdm2, c-jun and EGFR were up-regulated in lung squamous carcinomas, whereas they were down-regulated in adjacent normal bronchial epithelial tissues, normal lung tissues and inflammatory pseudotumor, which was consistent with our proteome analysis results. Conclusion： The well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were established and 68 differential proteins were characterized by applying comparative proteome analysis successfully. These results will provide scientific foundation for screening the molecular biomarker used to diagnose and treat lung squamous carcinoma, as well as to improve the patient＇s prognosis and provide new clue for the research of lung squamous carcinogenic mechanism.

Orthotopical transplantation of human renal carcinoma tissue into nude mice and the establishment of a high metastatic cell line MRCC

Objective To establish a SOI model of human renal carcinoma and a high metastatic cell subline. Methods A human renal cell line RCC-9863 has been established by inoculating a human renal tumor tissue into nude mice s. c.. When RCC-9863 passaged for 20 times, the tissue from the same xemotransplant tumor were used to construct SOI model. Cultured the metastatic tissue in vitro, the tumor cell suspension was then injected orthotopically, The metastatic tissue obtained underwent the same procedure again. At last, the metastatic tumor was cultured in vitro and cloned. Results 15 days later, a tumor mass sized 1. 7 cm × 0. 6 cm in the nude mouse’s renal parenchyma was grown which lobulated, rude, and with multiply blood vessels and 55 days later later the mouse became moribund and metastases in the lungs were formed. The transplanted renal tumor in the SOI model grew fast and invasively and metastasized to lungs, lymphatic node and liver. A subline, MRCC, with metastatic ability to the lung was selected.

VEGF in hepatocellular carcinoma and surrounding cirrhotic liver tissues

We read with a great interest the recent work of Deli and colleagues. in the World Journal of Gastroenterology reporting vascular endothelial growth factor （VEGF） expression in hepatocellular carcinoma （HCC） and cirrhotic liver tissues. This well-documented work shows that VEGF was significantly higher in surrounding cirrhotic liver tissues than in HCC. Authors assessed VEGF expression using immunohistochemistry. The immunohistochemical staining is an efficient tool to assess the percentage of cells stained positively for VEGF but is not really efficient to estimate their true VEGF content. Evaluation of the VEGF protein by an enzyme-linked immunosorbent assay 0ELISA） has been reported, by us and others, to be an efficient tool in order to assess tissue VEGF expression. We have, thus, tested whether the ELISA method might be an efficient tool in order to confirm data reporting higher amounts of VEGF in surrounding cirrhotic liver tissues than in HCC. Deli and colleagues. also correctly pointed out that basic fibroblast growth factor （bFGF） has been reported to act cooperatively on VEGF expression. We have, thus, also assessed bFGF tissue levels in order to search for a putative link between VEGF and bFGF levels in cirrhotic tissues.

THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

DIAGNOSTIC VALUE OF SERUM PANCREATIC TISSUE CARCINOMA ANTIGEN IN PANCREATIC CARCINOMA

Serum pancreatic tissue carcinoma antigen (PCA) was measured by ELISA in 396 patients with pancreatic carcinoma (PC) and other diseases diagnosed by clinical examination, histopathology and operation. Serum PCA more than 300 U/ml was considered as diagnostic value for PC and 30-299 U/ml as probable index for PC. The sensitivity of PCA for PC was 78.2% (43/55), and the specifity 94.6%. The positive rate of PCA in other diseases were 0-13.3%. Combining the determination of PCA with B-mode ultrasonic scanning (BU). CT and/or ERCP (endos-copic retrograde cholangiopancreatography) may increase the diagnostic rate of PC to 89-91%, and with the "cocktail" of CA19-9, CEA (carcino-embryonic antigen) and RNase-C, PC be diagnosed in 88.9%. Therefore, it is suggest that PCA si a promising tumor market of PC with high sensitivity and relative specificity.

Proteomic Comparison of Two-Dimensional Gel Electrophoresis Profiles from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorp-tion/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733±0.101 mm in IEF direction, and 0.925±0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190±72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduc-tion. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.