[Identification of metastasis-associated proteins of ovarian cancer by proteomics].

OBJECTIVE: To investigate the differenal protein expression profiles of ovarian tumor cell lines with distinct metastatic abilities. METHODS: The ovarian cancer cell line HO8910 and HO8910pm, derived from same parental cells but exhibited different metastatic ability, were investigated by two-dimensional gel electrophoresis (2-DE)-MALDI-TOF-MS proteomic approach. RESULTS: Thirty-nine proteins were detected by 2-DE to have expression disparity levels over 2 folds between two cell lines. Eighteen of them were identified by MALDI-TOF-MS. The proteins are involved in apoptosis, extra cellular matrix (ECM), cytoskeleton, growth factor, glycolysis, protein metabolism and immune system. CONCLUSION: The data are valuable for the identification of differentially expressed proteins involved in the biological behavior of human ovarian cancer.

Proteomics-based analysis of a pair of glioma cell lines with different tumor forming characteristics.

Glioma is the most common malignant disease in the brain, and recurrence is the main cause of death from this disease. Tumor recurrence involves multiple steps, and requires the accumulation of the altered expression of many different proteins. Identification of the recurrence associated protein profile in glioma cell lines will be helpful in clarifying the molecular mechanisms underlying glioma recurrence. In this report, two glioma cell lines with distinct tumor forming ability in vitro and in vivo were chosen and the different protein expression patterns were analyzed by proteomics method. To confirm the utility of this method, we validated the differential expression of one protein, cathepsin D, by immunohistochemistry analysis. Forty-six proteins appeared differently between two cell lines and 18 of them were identified. These 18 are involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis and neurotrophic factor. All of these molecules are important in tumor growth, and a subset of them may be related to glioma recurrence. These findings may contribute to the discovery of new diagnostic markers and therapeutic targets of glioma.

Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry.

AIM: To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOFMS). METHODS: The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software. RESULTS: PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified. CONCLUSION: The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

[Expression of IgVH and B7-1 in proteome of the human colorectal carcinoma cell lines].

OBJECTIVE: To conduct a proteomic analysis of human colorectal carcinoma cell lines LS174T and SW480 by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). METHODS: The total proteins of human colorectal carcinoma cell lines LS174T and SW480 were separated with 2-DE using immobilized pH gradient strips and analyzed by MALDI-TOF-MS to obtain peptide mass fingerprints (PMFs). Proteins were identified by using Mascot software to search protein databases. RESULTS: Good resolution 2-DE maps were obtained. Some proteins including immunoglobulin heavy chain variable region (IgVH) and co-stimulatory molecule B7-1 were identified. IgVH and B7-1 were confirmed by electrospray ionization tandem spectrometry (ESI-MS/MS) and immunocytochemistry. CONCLUSION: There are IgVH and B7-1 expressions in human colorectal carcinoma cell lines LS174T and SW480. Results obtained will help to elucidate the mechanisms of tumor immune escape.

[Poorly-immunogenic tumor is capable of inducing proliferation of CD4 (+) CD25 (+) regulatory T-lymphocytes in vitro].

OBJECTIVE: To investigate the distribution of regulatory T-lymphocytes in the splenocytes cocultured with syngeneic low-immunogenic tumor cells, as compared with that of highly-immunogenic tumor cells, to investigate the mechanism underlining tumor evasion. METHODS: Three different immunogenic tumor cells were cocultured with syngeneic splenocytes individually to mimic cancer immunity in vitro. The proliferation response of splenocytes was measured by thymidine incorporation. The distribution of TR cells, CD4(+) IFN-gamma (+) T cells and CD4(+) IL-10(+) T cells were analyzed by flow cytometry. The secretion of IFN-gamma and IL-10 in supernatants was measured by ELISA assay. RESULTS: The stimulation Index of splenocytes cocultured with syngeneic highly-immunogenic H22 or FBL3 was much higher than that of poorly immunogenic melanoma D5. In each group, stimulation Index of splenocytes cocultured with allogeneic tumor cells was higher than that of the corresponding tumor immunity model. In addition, compared with those of highly-immunogenic tumors, there were more TR, CD4(+)IL-10(+) and less CD4 (+)IFN-gamma(+) T cells in the splenocytes, and higher IL-10 and lower IFN-gamma levels in the supernatant of the splenocytes stimulated with low-immunogenic D5 cells. CONCLUSION: Poorly-immunogenic tumor cells can induce the proliferation of TR cells, which may play an important role in tumor evasion.

[Study of immune responses induced by human papillomavirus type 18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice].

AIM: To examine the humoral and cellular immunoresponses induced by HPV18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice. METHODS: 54 BALB/c mice were divided into 9 groups randomly, and then vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immune adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal). After the third inoculation, blood samples were taken to measure specific antibody, and footpad swelling test was used to detect delayed-type hypersensitivity(DTH). Mice were killed and spleens were taken to prepare single spleen cell suspension for lymphocyte proliferation assay and CD4(+)/CD8(+), IFN-gamma(+) or IL-4(+) T cells double staining FACS assay. And then the pathological changes of the swollen footpad were observed. RESULTS: Compared with control, significant immunoresponses were observed in different experimental groups. The level of specific serum IgG against HPV in experiment groups was much higher than that of control group, and intramuscular immunization group had the highest antibody level. The footpad from immunized mice injected with virus-like particles was swollen and harden, and a large amounts of mononuclear cells can be seen in the footpad tissues. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8(+) IFN-gamma(+) cell number, while CD4(+) IL-4(+) cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immune responses. CONCLUSION: The recombinant plasmids could induce strong humoral and cellular immunoresponses in mice. Costimulatory molecule B7-2 could enhance the immune responses against HPV18 induced by the L1-E6 and L1-E7 DNA vaccines when used as an adjuvant.

[Construction and expression of eukaryotic expression plasmid pcDNA3.1/hIL-18].

AIM: To construct an eukaryotic expression plasmid pcDNA3.1/hIL-18 and express it in mammalian cells. METHODS: cDNA encoding mature hIL-18 was cleavaged by enzyme digestion from mesomeric clone vector pGEM-T/hIL-18 and inserted into an eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/hIL-18. Then the constructed plasmid was transfected into COS-7 and Rlc310 cells by liposome-mediated gene transfer method. hIL-18 expressed in transfected COS-7 and Rlc310 cells was detected by immunohistochemical staining and level of hIL-18 mRNA in transfected Rlc310 cells was assayed by RT-PCR. RESULTS: A recombinant eukaryotic expression plasmid pcDNA3.1/hIL-18 was successfully constructed and expressed transiently in COS-7 cells and stably in Rlc310 cells. CONCLUSION: The construction and expression of pcDNA3.1/hIL-18 have been achieved successfully, which lays a foundation for further research on anti-tumor effect of IL-18.

[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471].

AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry. RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha. CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.