Proteome analysis and tissue array for profiling protein markers associated with type B thymoma subclassification / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The prognostic relevance of World Health Organization (WHO) subtypes within type B thymomas is still controversial. Understanding of the molecular characteristics of the different histologic types of thymomas will provide meaningful information for diagnosis and therapeutic management in type B thymoma.</p><p><b>METHODS</b>Proteins extracted from twelve type B thymoma tissue specimens (six type B1 and six type B2) were analyzed by two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF-MS. Differentially expressed proteins were then assayed in sixty-nine type B thymoma tissues (including B1, B2 and B3) by tissue array analysis with immunohistochemistry staining. The relationship of their expression with clinicopathological parameters, such as tumor stage or WHO classification, was estimated by Spearman's Rank Correlation Test.</p><p><b>RESULTS</b>Sixteen differentially expressed proteins between type B1 and B2 thymoma tissues were identified. The differential levels of ezrin and glutathione S-transferase pi (GSTP1) were validated using immunohistochemistry staining. A statistically significant difference was observed in the positive rate of ezrin expression between type B1 thymoma and type B3 thymoma (Z = -2.963, P < 0.01). Ezrin showed a tendency to be expressed in higher classification tumors from type B1 to B3. A statistical analysis demonstrated that type B2 and B3 tumors had significantly higher positive expression of GSTP1 than the B1 group (type B2 vs. B1: Z = -2.582, P = 0.01; type B3 vs. B1: Z = -4.012, P ≤ 0.001). The results also showed a strong correlation between GSTP1 and WHO type staging of B1 to B3 tumors (Spearman's correlation coefficient: 0.633, P ≤ 0.001). Statistical analysis showed that there was close correlation between GSTP1 and ezrin expression with the clinical stage (Spearman's correlation coefficients, ezrin: 0.481, P < 0.05; GSTP1: 0.484, P < 0.01).</p><p><b>CONCLUSIONS</b>Differentially expressed proteins between type B1 and B2 thymoma tissues were analyzed by comparative proteomic analysis. The techniques of proteomic analysis and tissue array provide a potential tool for screening of key molecules in type B thymoma histological sub-classifications. The statistical analysis of ezrin and GSTP1 expression by immunohistochemistry, especially GSTP1, may be a useful approach for type B thymoma classification.</p>

Cloning and expression of hTRF1 in Escherichia coli and preparation of polyclonal antibody / 生物工程学报

Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.