| Purpose 1) To observe the expression profile of MUC1 peptide andcarbohydrate epitopes in renal cell carcinoma (RCC) and bladder transitional cell carcinoma(TCC) . 2) To biopan phage displayed random peptide library to get peptides that mimic MUCl sialylated carbohydrate epitope and to preliminarily investigate the functional immunological responses induced with the mimotope and its effectiveness in prevention and treatment of MUC1-positive bladder TCC .Materials and Methods 1). The expression and distribution pattern of MUC1 carbohydrate and peptide epitopes were detected with immunohistochemistry, so as to their correlation with pathological grade and clinical stages. 2). Phage displayed random peptide library was biopanned to get 12-mer peptides that might mimic MUC1 sialylated carbohydrate epitope, which regcognized specifically by monoclonal antibody Ma695. Competitive inhibition assay by flowcytometry analysis with MUC1-positive tumor cell MCF7 was done to confirm the specificity of the peptide mimics. Dendritic cells were derived from mice mono-nuclear cells of splenocytes with GM-CSF and IL-4 and matured with LPS, the purity of DC was identified by FCM. Antibody responses cross-reacting with MUC1-positive tumor cells and mimotope-specific CTL response in mice immunized with mimotope-containing peptide pulsed DCs were studied with flowcytometry, ELISA and ELISPOT. In vitro CTL response was analyzed by non-radiative LDH cytotoxicity analysis. 3) Mice bladder TCC sub-cell line expressing human MUC1,designated BST739-MUC1, was established by Lipofectamine-mediated transfection of human MUC1 full length cDNA into BST739 cells, followed by G418selection and clonal culturing. PCR, RT-PCR, immunohistochemistry, confocal imaging, flowcytometry and Western blotting were carried out to confirm the stable integration of MUC 1 cDNA into the mouse genome and expression of MUCl mRNA and proteins in the cells. The in vitro adhesive and growth characteristics of BST739-MUC1, as well as its in vivo tumorigenicity were also investigated. 4) The preventive and therapeutic mice models of bladder TCC were established to investigate preliminarily the effectiveness of MUCl mimotope-containing peptide as a tumor vaccine in the inhibition of MUCl-positive tumor cells growth in vivo. Results 1) The expression of MUClwas detected in 85.5% of RCCand 100% of TCC. The percentage of positive cells and their distribution model were closely correlated with pathological grade and clinical stages. 2) Ml-peptide that mimic MUCl carbohydrate epitope was obtained through a 3-round biopanning of phage displayed random 12-mer peptide library. Ml inhibited the binding of McAb Ma695 to MCF7 competitively. As the cell number (2xlO5) and the concentration of Ma695(0.25ug/ml) were fixed, when the concentration of Ml was 100 ug/ml ,200 ug/ml, 400 ug/ml and 800 ug/ml, the inhibition rate was 11%, 26%, 26% and 34%, respectively. After a 6-day culture and 1-day maturation with LPS, cells with characteristic appearance of mature DC were harvested, FCM showed that 83.2% cells expressed a marker of mature DC. Ml-specific CTL could be induced with Ml-pulsed DC, as the results of ELISPOT showed that the frequency of functional T cells (IFN-ysecreting lymphocytes determined in ELISPOT) in immunized mice was 116/105 spleen cells. Also, specific antibody responses induced by Ml-pulsed DC, which could cross-react with MUCl carbohydrate epitope, were detected and the FCM results showed that serum from immunized mice could react with MUC 1-positive tumor cells, but not MUCl-negtive ones. 3) The MUCl gene integration, transcription and protein expression in BST739-MUC1 were confirmed bygenomic DNA PCR, RT-PCR, immunohistochemistry and Western-blotting, Confocal imaging indicated that MUCl protein expressed in the cell membranes as well as in the cytoplasm. Compared with BST739, the in vitro adhesion rate of BST739-MUC1 increased by 44% through MTT assay. The syngeneic mice would reject MUCl-positive tumor cells, for this reason, the in vivo growth of BST739-MUC1 had a characteristic of fast-slow-fast fashion. During the first 18 days after inoculation, the growing speed of BST739-MUC1 was much faster. On the 18th day, the volume of BST739-MUC1 tumor was 2744±948mm3 vs 808±322mm\ p=0.0013) , and then the growth rate was slowed down during the 18th to 26th day and from the 26th day on, the growth rate accelerated again. 4). Splenocytes of mice immunized with Ml-pulsed DC showed antitumor reactivity in vitro, as determined by CTL analysis. The splenocytes could kill 18.9%~29.5% BST739-MUC1 when the E/T ratio was 100:1. In the preventive model, tumor weight of mice immunized with Ml-pulse DC was only 14.4%(1.7±0.8mg vs 11.8±4.8 mg, p O.001) of that in the control group. Ml-reacting antibody concentration increased from 0.57±0.21 to 1.52±0.54(167% increase)and CTL reactivity increased by 122%(17.3% vs 7.8%). In the therapeutic model, the life duration of mice challenged with 2xlO6 BST739-MUC1 and then treated with Ml-pulse DC prolonged 50%(44±5.7days vs 30±1.6days, p=0.0031) , the concentration of Ml-reacting antibody showed a 338% (1.71 vs 0.39) increase and a 71% (19.5% vs 11.4%) increase of the CTL reactivity was also observed. Conclusions 1) Over-expression and aberrant distribution of MUCl were common in RCC and TCC, indicating that MUCl was an ideal target for antitumor vaccine of these malignant tumors. 2) Mice bladder cancer cell line BST739-MUC1 stably expressed human MUCl and BST739-MUC1 -associated tumor models were established. 3) 12-mer peptide Ml that mimic MUCl carbohydrate epitope was identified and cellular and...
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