| The 22-1-1 MoAb was generated by immunizing BALB/c mice with the human uterine cervical adenocarcinoma cell line, SiSo, which was constructed by SONODA et al (cancer, 1996), and the molecule weight of the 22-1-1 Ag was 40kD and 78kD, the latter which was thought to be the homodimer of the former. It was considered as a novel cancer associated antigen recognized by 22-1-1 MoAb. In 1998, an estrogen-responsive gene, named EBAG9, was isolated from the cDNA library of human breast cancer cell line, MCF-7, and the estrogen responsive element (ERE) was located in a 5' upstream position of the cDNA (Mol Cell Biol, 1998). The cDNA had a coding region of 639 nucleotides, and its expression was upregulated by estrogen both in the level of mRNA and protein. NAKASHIMA et al (NatMed, 1999) identified the gene encoding 22-1-1 Ag and was defined as RCAS1 (receptor-binding cancer antigen expressed on SiSo cells), and was considered as the same protein as EBAG9. In their opinion, RCAS1 was a type II membrane protein with an N-terminal transmembrane segment and a coiled-coil structure in the C-terminal portion. Their finding indicated that RCAS1 acted as a ligand for a putative receptor present on various human cell lines and normal peripheral lymphocytes such as T, B and NK cells, and therebytumor cells might evade immune surveillance by expression of RCAS1. The receptor of RCAS1 had been detected on the K562 cells and the binding of RCASl with the receptor stimulated the activation of caspases 8 followed by the development of apoptosis. However, ENGELSBERG et al (J Biol Chem, 2003) thought that RCASl was a ubiquitously expressed type III transmembrane protein with a Golgi-predominant localization. Their finding showed that the cognate 22-1-1 epitope was identical with the tumor-associated O-linked glycan Tn, and the 22-1-1 MoAb failed to recognize RCAS1. Overexpression of RCASl in cell lines that were negative for 22-1-1 surface staining led to the generation of Tn and the closely related TF (Thomsen-friedenreich, Galβ-3GalNAc). About RCASl, most the publication used immunohistochemical analysis of the expression of this molecule on various tumors or cancers such as breast cancer, lung adenocarcinomas, gastric carcinoma et al. RCAS1 was considered as a marker correlated with poor dedifferentiation, invasion, poor prognosis. However, these findings were all based on the premise: 22-1-1 Ag was identical to RCAS1 (or EBAG9) , because the immunohistochemical analysis wwas performed by 22-1-1 MoAb.Based on the above progress in the field of RCASl research, the goal of our study was to clarify the function of RCASl in induction of immune Cell hyporesponsiveness and its related mechanism. This study was divided into four parts. Firstly, we tried to verify whether 22-1-1 Ag was identical to the antigen RCASl (or EBAG9), because the correlation between tumor development and expression of RCASl was analyzed by 22-1-1 monoclonal antibody. So the construction of GST-RCAS1 fusion protein and the identification by specific antibody: 22-1-1 MoAb and RCASl polyclonal antibody were necessary for this project. And the fusion protein GST-RCAS1 was an effective tool for the study of the function of RCASl.tn fM/ainnto tVi*? fiinftirtri nfPPACI am ticoA +Vw? fi.o^n nmtom nOT DPAC1and advenovirus vector of RCASl (Ad-RCASl), to investigate apoptosis of immune cells induced by RCASl. Thirdly, we investigated the regulatory effect of estrogen and progesterone on the expression of RCASl gene in human breast cancer cell line MCF-7. Lastly, many literatures showed tumor metastases were related with the expression of RCASl, but no experimental data (in vivo or in vitro) were shown to demonstrate it. We generated the 4T1/RCAS1 cell line which stably overexpresses RCASl, and synthesized RCASl siRNA which could effectively knockdown the RCASl expression. A metastatic breast cancer model was established in BALB/c mice and the effect for RCASl overexpression and knockdown on the tumor growth and lung metastases was investigated.Part I Construction of Recombinant Plasmid of RCASl and the Expression, Purification, and Identification of GST-RCAS1 Fusion ProteinObjectives: To construct the recombinant plasmid of a novel tumor associated antigen, RCASl, to express and purify its fusion protein, GST-RCAS1, and to identify it by western blotting.Methods: RCASl encoding gene was amplified by RT-PCR from total RNA extracted from MCF-7 cells and was ligated with expression plasmid vector pGEX-2T by T4 DNA ligase after digested by the restricted endonucleases, BamHI and EcoRI. Then the ligated products were inserted into competence JM109 E. coli and the positive recombinants were identified by restriction endonuclease digestion assay and DNA sequencing. The GST-RCAS1 fusion protein expression was induced by IPTG in BL21 E. coli and was purified with GST column and identified by SDS-PAGE and Western blotting with anti-GST monoclonal antibody, anti-22-1-1 monoclonal antibody, anti-RCASl (N-18) and anti-RCASl (C-20) polyclonal antibody.Results: A 642bp product was cloned by RT-PCR and the recombinant plasmid wasconstructed successfully. The GST-RCASl fusion protein could be recognized by both GST monoclonal and the RCASl (N-18 and C-20) polyclonal antibody. Unexpectedly, the fusion protein were failed to be recognized by 22-1-1 MoAb.Conclusions: The recombinant plasmid of RCAS1 was successfully constructed and the GST-RCASl fusion protein was expressed, purified, and identified. It showed that 22-1-1 Ag was not the same protein as RCASl or EBAG9.Partll The Role of RCASl in inducing apoptosis of immune cells Ojectives: To study the biological function of RCASl in vitro in inducing apoptosis of immune cells, to investigate the role of RCASl in inhibiting cell growth, to detect the receptor of RCASl on relative cells, and to analyze the related mechanism of apoptosis induced by RCASl.Methods: 1. Identification of the recombinant Ad-RCASl. Hela cells were infected with either Ad-RCASl or Ad-LacZ, and the total RNA was extracted for RT-PCR;total protein was extracted for western blotting (22-1-1 MoAb and RCASl polyclonal antibody were used). 2. Detection of apoptosis in T lymhocytes and immune cell derived leukemia cell lines after treatment of RCASl. The mononuclear cells were obtained from peripheral blood of health donors and the apoptosis in T lymphocytes and K562, Jurkat, THPl and E003 cells after treated with GST-RCASl or Ad-RCASl were detected by Annexin V and PI staining using FACS. 3. Cell growth inhibition by RCASl. MTT assay was used to evaluate the inhibition effect of RCASl on the growth of Jurkat and K 562 cells. 4. RCASl receptor detection. Putative RCASl receptor was measured in Jurkat and K562 cells, hi brief, the cells were co-incubated with GST-RCASl fusion protein followed washing three times with PBS. Then the GST MoAb was added and followed the staining of anti-IgG conjugated FITC, after washing three times, the cells was detected by FACSand the FITC positive cells was measured. 5. The relationship between overexpression of RCASl and GSK30 or phGSK3p. K562 cells were infected with Ad-RCASl or Ad-LacZ (as a control) with Moi 0, 10, 20, 40, 80, 160, and western blot was used to detect the changes of RCASl and GSK3|3or phGSK3|}expression. Co-immuoprecipitation was used to analyze the binding of RCASl and GSK3p.Results: 1. Semi-quantitiative RT-PCR analysis showed that the mRNA expression of RCASl in Hela cells infected with Ad-RCASl was higher than Hela cells infected with Ad-LacZ or parental Hela cells. Simultaneously, the density of the 32 kD bands detected by WB using polyclonal antibody RCASl C20 and N18 was relatively higher in the cell lysates of Hela cells infected with Ad-RCASl than that in Hela cells infected with Ad-LacZ or parental Hela cells. 2. Activated T cells could be induced to undergo apoptosis by either GST-RCAS1 or Ad-RCASl, but Ad-RCASl played more potent role in inducing apoptosis. In cell lines Jurkat, K562, THP1 and E003, apoptosis could be detected after Ad-RCASl, but in the protein group, the results were not so consentaneous. 3. In the adenovirus group with the MOI from 12.5 to 100, MTT results showed that RCASl inhibited the cell growth of both Jurkat cells and K562 cells. However, the fusion protein GST-RCAS1 played no role in inhibiting cell growth. 4. In K562 cells, RCASl receptor could be detected and the percentage of FITC positive cells was not high, but statistical difference did exist. As to Jurkat cells, no FITC positive cells could be detected, but after the cells were treated with PHA with the final concentration of lug/ml, markedly higher percentage of FITC positive cells were detected (43%). 5. With the increase of RCASl expression in k562 cells, the expression of GSK3P was downregulated whereas phGSK3p increased. However, the co-immunoprecipitation showed no binding between RCASl and GSK30 or phGSK3p\ But the result of co-immunoprecipitation also showed that the protein immunoprecipitated by RCAS1/C20 polyclonal antibody could not be recognized bythe 22-1-1 monoclonal antibody, which further demonstrated that the 22-1-1 Ag was not the same protein as the RC AS 1.Conclusions: l.HeLa cells infected with Ad-RCASl could highly express RCASl in mRNA and protein level. It was confirmed that 22-1-1 Ag was not the same protein as RCASl. 2. RCASl could induce apoptosis of activated T cells, K562 cells and PHA activated Jurkat cells via the RCAS1-RCAS1R pathway. 3. RCASl receptor could be induced to express. 4. The intracellular overexpression of RCASl could inhibit the cell growth of Jurkat and K562 cells. 5. GSK3p may be involved in the pathway through which RCASl induces apoptosis of immune cells.Partm Effect of estrogen and progesterone on the expression of RCASl in human breast cancer cell line MCF-7Objectives: To study the eflfect of estrogen (17P-estradiol) and progesterone on expression of RCASl in breast cancer cell line MCF-7 in vitro. Methods: 1.dose-dependent assay. Human breast cancer cell line MCF-7 cells were exposed to estrogen (10"12~10"8M) or progesterone (10"9~10*5M) for 72h, and the expression level of RCASl mRNA was determined by RT-PCR, then western blot assay was used to analyze the change of RCASl protein expression. 2. Time-dependent assay. MCF-7 cells were treated with estrogen (10'10M) or progesterone (10'8M) from 12h to 72h and harvested every 12h. RT-PCR and western blot were used to determine the expression of RCASl in the level of mRNA and protein. 3. MTT assay. This experiment was used to evaluate the growth inhibition effect of estrogen or progeseterone on the MCF-7.Results: The PCR products of RCASl were 642bp. The expression of RCASl mRNA in the estrogen-treated cells was significantly higher than that in the untreated controls (p<0.05), and the expression level of RCASl mRNA was upregulated with theincrease in estrogen concentration or with the increase in the estrogen exposure time. The expression of RCAS1 mRNA in MCF-7 treated with progesterone was significantly lower than that in the untreated control (p<0.05), and the expression of RCAS1 mRNA was downregulated with the increase in progesterone concentration or with the increase in the progesterone exposure time. A 32kD band was detected in MCF-7 cell extracts by western blot assay and, with the increase of estrogen or progesterone concentration, or with the increase exposure time in either estrogen or progesterone, the bands were detected at a higher or lower intensity compared to untreated control. In MTT assay, no growth inhibition effect was detected in MCF-7 cells treated with estrogen, whereas high dose progesterone played a slightly inhibiting effect on MCF-7 cell proliferation.Conclusions: Estrogen could up-regulate, while progesterone could down-regulate the expression of RCAS1 both in the level of mRNA and protein with the dose- and time-dependent manner.Part IV Study on the relationship between expression of RCAS1 and tumor growth, development, and metastases of murine 4T1 breast cancer Objectives: To generate 4T1 cell line stably overexpressing mRCASl(mouse RCAS1), 4Tl/mRCASl, to design and screen an effective siRNA to silence the expression of mRCASl in 4T1 cells, and to reveal the relationship between RCAS1 expression and tumor growth, development, and lung metastases of 4T1 tumor. Methods: 1. Generation of 4T1 cells stably overexpressing mRCASl. mRCASl encoding gene was amplified by RT-PCR from total RNA extracted from 4T1 cells and was ligated with expression plasmid vector pcDNA3.1 by T4 DNA ligase after digested by the restricted endonucleases, EcoRI and BamHI. The recombinants were identified by restriction endonuclease digestion assay, PCR, and DNA sequencing.4T1 cells were transfected with plasmid pcDNA3.1 /mRCASl, using lipofectAMINE. G418 resistant cells were selected and several independent clones were isolated. After the transfected cells 4T1/mRCASl and 4Tl/mock was stably constructed, RT-PCR and western blot were used to determine the expression of mRCASl (parental 4T1 as a control). 2. The design and screening of siRNA. According to the mRCASl sequence, three siRNAs were designed and generated to silence the mRCASl gene in 4T1 cells. A nonsense siRNA (as negative control) and a GAPDH siRNA (as positive control) were also generated to demonstrate the effectiveness of the screening assay. The effective siRNA were screened by RT-PCR and western blot. 3. Cell proliferation assay. To determine whether the recombinant plasmid pcDNA3.1/mRCASl and RCAS1 siRNA2 influenced the growth of 4T1 cells in vitro, apoptosis was detected by FACS and MTT assay were used. 4. The construction of 4T1 tumor models and the treatment of the experimental groups. 4T1, 4T1/mRCASl, and 4Tl/mock cells were collected and prepared to single cell suspension with lxlO7 cells/ml. Female BALB/c mice, 6~8 weeks old, were divided into five groups (6 mice in each group). Mice in the first to the third group (4Tl/control, 4Tl/siRNA, and 4T1/Nonsilencing, respectively) were subcutaneously injected with l*106 4T1 cells, whereas mice in the forth or fifth group were subcutaneously injected with l*106 4Tl/mRCASl or 4Tl/mock cells, respectively. Three day later after the tumor inoculation the tumor size was measured with a micrometer in two dimensions every other day. Five days after the inoculation siRNA2 (lOug) or Nonsilencing siRNA (10|ag) was injected intratumorally in mice of the second or third groups every four days, along with 4ul Genesilencer dissolved in lOOul of Opti-MEM. All mice were sacrificed 1 month after treatment. 4. Detection of tumor growth and metastasis. Tumor volume were calculated with the formula: V =longest diameterxsmallest diameter2, and statistically analyzed by student / test. Allmice were sacrificed a month after tumor inoculation, part of spienocytes were used directly to measure NK (target cell: YAC-1) activity. The other spienocytes of three mice in every group were stimulated for 5 days with mitomycin treated 4T1 cells and specific (target cell: 4T1) CTL activity were measured by LDH release assay with CT26 as a control target cells. To analyze lung tumor metastasis, the lungs of three sacrificed mice were removed and injected with India ink to visualize individual tumor nodules. Then the other lungs from the sacrificed mice were used to perform colony formation assay. In brief, lungs were minced and single cell suspensions were cultured in a medium contaning 6-thioguanine (60umol/L) for colony formation. Tumor tissues were removed from mice for RT-PCR and western blot to determine the expression of RCAS1 in every group, and for HE staining. The whole in vivo experiments were repeated twice.Results: 1. Expression vector of mRCASl was constructed. A 642bp product was cloned by RT-PCR and the recombinant plasmid pcDNA3.1/mRCASl was identified by restriction enzyme digestion, PCR and sequencing. 2. By G418 resistant screening, 4Tl/mRCASl and 4T1/Mock cell lines were constructed successfully and the RCAS1 expression was verified by RT-PCR and western blotting. 3. Among the three siRNAs, siRNA2 effectively silenced the expression of mRCASl in 4T1 cells. 4. 4T1 cell viability was not affected by transfection either with pcDNA3.1/RCASl or RCAS1 siRNA2. 5. Average tumor volume of mice in 4Tl/siRNA2 group or in 4T1/Mock group was significantly smaller than that in 4T1/Nonsilencing group or 4T1/RCAS1 group. No statistical difference was shown among the 4T1/Control, 4T1/Nonsilencing and 4T1/RCAS1 group. 6. No significant difference was detected among the five groups in NK activity. Specific CTL cytotoxicity assay showed that splenic CTL activity in 4Tl/siRNA2 group was significant higher than that in other four groups. There was no difference among the 4T1/Control, 4T1/RCAS1 and 4T1/Mock group. 7.Lung staining assay using India Ink showed that the number of metastatic tumor nodules in lungs from 4T1/RCAS1 group was significantly more than that from other four groups (p<0.05). Lung colony formation assay showed that the number of colonies per lung from 4T1 /RCASlgroup were significantly more than that from other groups (p<0.0\), and the number of colonies from 4Tl/siRNA group was significantly less than the other groups (p<0.05). 8. RT-PCR and western blot assays demonstrated the overexpression of RCASl in the 4T1/RCAS1 tumors and very low expression of RCASl in tumors after 4Tl/siRNA2 treatment. 9. More infiltration of inflammatory cells including lymphocytes inside the tumors was observed in tumor tissue from RCAS1 siRNA treated mice.Conclusions: Overexpression of RCASl in 4T1 cells could promote tumor growth and metastasis, and RCASl siRNA was shown to effectively inhibit the tumor growth and lung metastasis.Summary1. Recombinant plasmid pGEX-2T/RCASl was successfully constructed and GST-RCAS1 fusion protein was expressed, purified and identified. Western blot using 22-1-1 Ab, RCASl Abs suggested that 22-1-1 Ag was not the same protein as RCASl.2. RCASl could induce apoptosis of activated T cells, K562 and activated Jurkat leukemia cell lines. A putative RCASl receptor was detected in K562 cells and activated Jurkat cells after treatment of PHA, indicating RCASl induces apoptosis through binding of its receptor. Overexpression of RCASl might be related with phosphorylation of GSK3|3and followed by the inhibition of NF-kB pathway.3. 17f}-estradiol could up-regulate, while progesterone could down-regulate the expression of RCASl both in the level of mRNA and protein with the dose- andtime-dependent pattern.4. In vivo experiment showed that overexpression of RCASl was correlated with tumor growth and metastases in murine 4T1 breast cancer. And knockdown of the RCASl expression by small interfering RNA effectively inhibited the tumor growth and lung metastases, splenic CTL activity was markedly induced in mice received siRNA therapy, and more infiltration of inflammatory cells including lymphocytes inside the tumors were observed in tumor tissue from RCASl siRNA treated mice. These results will likely provide important insights to the mechanisms through which tumors evade the immunosurveillance and might provide a novel strategy for therapeutic intervention of cancer and tumor metastases.
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