TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning

The identification of novel tumor antigens is of extreme importance for effective immunotherapy against cancer. A major obstacle in this field is the limited life span of tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. Therefore we searched for a method to isolate the tumor specificity of these CTLs, i.e., their T-cell receptors (TCRs) and transfer it to an immortalized T-cell line. For this purpose, a TCR-negative Jurkat T-cell line was equipped with a nuclear factor of activated T cells (NFAT)-luciferase reporter construct to allow measurement of TCR-mediated activation. To establish the feasibility of this tumor-specific TCR transduction, we cloned the TCR genes of a known T-cell clone specific for the tumor antigen CAMEL (CTL-recognized antigen on melanoma) into a retroviral construct. Jurkat reporter cells transduced with this construct, Jrt-TCRalpha3beta5, were tested for their reactivity against CAMEL-expressing melanoma cells, peptide-loaded T2 cells and CAMEL-transfected COS-1 cells. The melanoma cell lines were poorly recognized, but peptide-pulsed and -transfected cells effectively stimulated NFAT signaling. The activation of TCR(+) Jurkat reporter cells was shown to be dependent on the antigen density on the target cells and the expression level of the coreceptor CD8 on the Jurkat cells. To verify the benefit of this TCR reconstitution method for identification of novel antigens, pools of the cDNA library from which CAMEL was originally cloned were transfected in COS-1 cells and screened with Jrt-TCRalpha3beta5. Identical cDNA pools were found that were positive with these cells and with the CAMEL-specific CTL clone. Our results illustrate that TCR-reconstituted Jurkat reporter cells are a useful tool in the identification of novel tumor antigens by cDNA expression cloning.     

Modulating metastasis by a lymphangiogenic switch in prostate cancer

Prostate cancer dissemination is difficult to detect in the clinic, and few treatment options exist for patients with advanced-stage disease. Our aim was to investigate the role of tumor lymphangiogenesis during metastasis. Further, we implemented a noninvasive molecular imaging technique to facilitate the assessment of the metastatic process. The metastatic potentials of several human prostate cancer xenograft models, LAPC-4, LAPC-9, PC3 and CWR22Rv-1 were compared. The cells were labeled with luciferase, a bioluminescence imaging reporter gene, to enable optical imaging. After tumor implantation the animals were examined weekly during several months for the appearance of metastases. Metastatic lesions were confirmed by immunohistochemistry. Additionally, the angiogenic and lymphangiogenic profiles of the tumors were characterized. To confirm the role of lymphangiogenesis in mediating metastasis, the low-metastatic LAPC-9 tumor cells were engineered to overexpress VEGF-C, and the development of metastases was evaluated. Our results show CWR22Rv-1 and PC3 tumor cell lines to be more metastatic than LAPC-4, which in turn disseminates more readily than LAPC-9. The difference in metastatic potential correlated with the endogenous production levels of lymphangiogenic growth factor VEGF-C and the presence of tumor lymphatics. In agreement, induced overexpression of VEGF-C in LAPC-9 enhanced tumor lymphangiogenesis leading to the development of metastatic lesions. Taken together, our studies, based on a molecular imaging approach for semiquantitative detection of micrometastases, point to an important role of tumor lymphatics in the metastatic process of human prostate cancer. In particular, VEGF-C seems to play a key role in prostate cancer metastasis.     

Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer

BACKGROUND: Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. METHODS: LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. RESULTS: The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. CONCLUSIONS: The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo.     

MDR1启动子/荧光素酶质粒的构建及其在朊蛋白高表达胃癌细胞中的活性检测

目的:构建含MDR1基因启动子的荧光素酶报告基因质粒,并检测其在朊蛋白高表达胃癌细胞系中的活性表达。方法:PCR克隆人MDRl基因启动子片段,通过亚克隆将启动子分别插入到pMD18-T载体和荧光素酶报告基因pGL3-Enhancer载体中,建立含MDR1启动子的荧光素酶报告基因质粒pGL-MDR1,并经测序及酶切确定扩增序列;脂质体基因转染法将pGL-MDR1转染入朊蛋白高表达胃癌细胞系SGC7901-PrP,并测定其荧光素酶活性。结果:PCR克隆出MDR1启动子经DNA测序证实序列正确,pGL-MDR1转染入朊蛋白高表达胃癌细胞系的荧光素酶活性,较转染入pcDNA3.1空载体细胞系相比升高3~5倍。结论:成功构建含MDRl启动子的荧光素酶报告基因质粒;上调朊蛋白表达可激活MDR1的转录活性。     

HIV-1 Tat蛋白结合Tar的抗艾滋病药物筛选模型的建立研究

目的建立HIV-1Tat蛋白结合Tar的抗HIV-1药物筛选模型用于抗HIV药物筛选。方法构建表达HIV-1Tat蛋白的真核表达质粒（pCDNA3，1（＋）-Tat）和HIV-1 LTR-luc荧光素酶报告基因，采用脂质体转染法共转染HeLa细胞，荧光仪检测Tat蛋白促进荧光素酶在HeLa细胞内的表达情况，建立HIV-1Tat蛋白结合Tar的细胞模型，用于抗HIV-1的药物筛选。以DRB（5，6-二氯-1-β-呋核亚硝脲-苯并咪唑）为阳性对照，进行模型的建立及条件的优化。结果通过反复试验表明，目的基因和报告基因的配比、转染时培养液中小牛血清的含量、细胞浓度、转染前细胞状态以及药物作用时间的长短等对模型的稳定性和敏感性有影响。结论应用优化的细胞模型对文冠果、紫苏等浸提物进行筛选及结果的分析。     

Cadmium-induced postaxial forelimb ectrodactyly: association with altered sonic hedgehog signaling

Administration of CdSO₄ to C57BL/6 mice at day 9.5 of gestation induces a high incidence of postaxial forelimb ectrodactyly in the offspring. We propose that Cd²⁺ exposure impairs the process of anterior/posterior formation in the limb bud, a process that is directed by Sonic hedgehog (Shh) signaling. We show that exposure of the mouse embryo to Cd²⁺ disrupts Shh signaling as measured by polarizing activity of mouse limb bud ZPA grafted to a host chick wing, and activity of a Gli:luciferase reporter exposed to limb bud lysates. Yet the expression of Shh and its translation are not affected by Cd²⁺ exposure. We propose that teratogen exposure affects the processing of Shh in the cells in which it is made.     

基于荧光素酶生物发光检测方法的研究及其应用进展

生物发光现象广泛存在于自然界中,不管是在陆地或是海洋都有发光生物的踪迹。其中基于荧光素酶的生物发光系统被广泛研究,启发着人类在基因、表观遗传等方面进行探索,并且开发出一系列相关的检测方法,用于体内体外各方面研究。本文从生物发光系统、荧光素酶的种类以及荧光素酶生物发光检测方法的开发与应用这几个方面进行总结,简要概括近年来基于荧光素酶生物发光检测的研究进展。     

Imaging CD8+ T cell dynamics in vivo using a transgenic luciferase reporter

After activation, populations of antigen-specific T cells flow between sites of antigen expression, local lymphoid structures and other lymphoid and non-lymphoid organs. In this study, we documented the in vivo dynamics of a CD8(+) T cell response to antigen delivered using herpes simplex virus amplicon vectors and revealed several unexpected features. First, the T cells localized to the site of vector injection, as well as the draining lymph node within 24-48 h. Second, the major site to which T cells later redistributed were intra-abdominal lymphoid organs, including milky spots, mesenteric and lumbar lymph nodes. We determined the relationship between bioluminescent signal and antigen-specific T cell numbers in various lymphoid organs, and concluded that bioluminescent signal is a valid surrogate measure of T cell abundance in superficial lymph nodes, but not in deeper structures such as the spleen.     

Interaction between the insulin receptor and Grb14: a dynamic study in living cells using BRET

Grb14 is a molecular adaptor that binds to the activated insulin receptor (IR) and negatively regulates insulin signaling. We have studied the dynamics of interaction of the IR with Grb14, in real time, in living HEK cells, using bioluminescence resonance energy transfer (BRET). Insulin rapidly and dose-dependently stimulated this interaction. Removing insulin from the incubation medium only resulted in a modest decrease in BRET signal, indicating that the interaction between the IR and Grb14 can remain long after insulin stimulus has disappeared. BRET saturation experiments indicated that insulin markedly increases the affinity between IR and Grb14, resulting in recruitment of the adaptor to the activated IR. In addition, using both BRET and co-immunoprecipitation experiments, we demonstrated that insulin induced the dimerization of Grb14, most likely as a result of simultaneous binding of two Grb14 molecules on the activated IR. We also investigated the relationships between IR, Grb14 and the protein tyrosine phosphatase PTP1B. We observed that insulin-induced BRET between the IR and PTP1B was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the insulin receptor, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the ERK pathway. Our work suggests that Grb14 may regulate signalling through the insulin receptor by controlling its tyrosine-dephosphorylation in a site-specific manner.