CEA Gene Research Articles

Two waves of evolution in the rodent pregnancy-specific glycoprotein (Psg) gene family lead to structurally diverse PSGs

BackgroundThe evolution of pregnancy-specific glycoprotein (PSG) genes within the CEA gene family of primates correlates with the evolution of hemochorial placentation about 45 Myr ago. Thus, we hypothesized that hemochorial placentation with intimate contact between fetal cells and maternal immune cells favors the evolution and expansion of PSGs. With only a few exceptions, all rodents have hemochorial placentas thus the question arises whether Psgs evolved in all rodent genera.ResultsIn the analysis of 94 rodent species from 4 suborders, we identified Psg genes only in the suborder Myomorpha in three families (characteristic species in brackets), namely Muridae (mouse), Cricetidae (hamster) and Nesomyidae (giant pouched rat). All Psgs are located, as previously described for mouse and rat, in a region of the genome separated from the Cea gene family locus by several megabases, further referred to as the rodent Psg locus. In the suborders Castorimorpha (beaver), Hystricognatha (guinea pig) and Sciuromorpha (squirrel), neither Psg genes nor so called CEA-related cell adhesion molecule (Ceacam) genes were found in the Psg locus. There was even no evidence for the existence of Psgs in any other genomic region. In contrast to the Psg-harboring rodent species, which do not have activating CEACAMs, we were able to identify Ceacam genes encoding activating CEACAMs in all other rodents studied. In the Psg locus, there are genes encoding three structurally distinct CEACAM/PSGs: (i) CEACAMs composed of one N- and one A2-type domain (CEACAM9, CEACAM15), (ii) composed of two N domains (CEACAM11-CEACAM14) and (iii) composed of three to eight N domains and one A2 domain (PSGs). All of them were found to be secreted glycoproteins preferentially expressed by trophoblast cells, thus they should be considered as PSGs.ConclusionIn rodents Psg genes evolved only recently in the suborder Myomorpha shortly upon their most recent common ancestor (MRCA) has coopted the retroviral genes syncytin-A and syncytin-B which enabled the evolution of the three-layered trophoblast. The expansion of Psgs is limited to the Psg locus most likely after a translocation of a CEA-related gene – possibly encoding an ITAM harboring CEACAM. According to the expression pattern two waves of gene amplification occurred, coding for structurally different PSGs.

Novel cloth-based closed bipolar solid-state electrochemiluminescence (CBP-SS-ECL) aptasensor for detecting carcinoembryonic antigen

This study involved the construction of a novel cloth-based CBP-SS-ECL aptasensor by introducing proximity hybridisation (PH) and solid-state electrochemiluminescence (SS-ECL) into a closed bipolar (CBP) electrochemistry system. Screen-printing was used to fabricate the cloth-based aptasensors, and PH and aptamers provided good selectivity for the sensor. Furthermore, the CBP-SS-ECL sensing interface was produced by immobilising of Ru(bpy)32+- doped silica nanoparticles (Ru–Si-NPs) on the anode of closed bipolar electrode (C-BPE). PH product (PP) was formed due to the target (carcinoembryonic antigen, CEA), and then the PP opened the Au NP-labelled capture probe (Au-CP) on the C-BPE anode (CBA), allowing ferrocene carboxylic acid (FcA) close to the CBA surface and Au NPs away from the surface. The corresponding quenching and enhancement of ECL signal by FcA and Au NPs, respectively, markedly enhanced ECL signal. This aptasensor successfully achieved quantitative detection of CEA, with a linear detection range of 5–100000 pg mL−1 and a detection limit of 1.6 pg mL−1. The described aptasensor showed an acceptable sensitivity, selectivity, stability, and ability to detect CEA levels in complex human serum samples. In particular, this aptasensor showed satisfactory versatility, indicating its potential for simultaneous detection of CEA and K-ras gene.

Effect of extracellular vesicles of Lactobacillus rhamnosus GG on the expression of CEA gene and protein released by colorectal cancer cells.

Background and Objectives:Almost all living cells secret nano-sized structures enclosed by the lipid bilayer called extracellular vesicles (EVs) into their extracellular milieu. These EVs play important roles in several physiological processes as a cargo delivery system. In probiotics, EVs are the main communication tool with the host. The present study aimed to assess the effect of EVs originated from Lactobacillus rhamnosus GG on the Carcinoembryonic antigen (cea) gene expression and protein (CEA) synthesis in the SW480 and HT-29 cell lines.Materials and Methods:Different concentrations of Lactobacillus rhamnosus GG EVs were applied on the SW480 and HT-29 cell lines. The MTT assay, Real-Time PCR, and ELISA analysis methods were exploited to explore the cell viability and the expression level of the cea gene in comparison with the β-actin gene as the control.Results:The two concentrations of 80 and 100 μg/ml of Lactobacillus rhamnosus GG EVs considerably affected the anti-proliferation and increased the amount of both CEA mRNA and protein (p < 0.05).Conclusion:Our findings showed that EVs of Lactobacillus rhamnosus GG could induce the gene expression and protein synthesis of CEA. Also, they reduced the cell proliferation of HT29 and SW480. Thus, probiotics such as EVs of Lactobacillus rhamnosus GG could be useful for preventing colorectal cancer.

A phase I, open-label, multicenter, single-dose escalation and multi-dose study of a monoclonal antibody targeting CEACAM1 in subjects with selected advanced or recurrent malignancies.

3094 Background: The carcinomembryonic antigen cell adhesion molecule 1 (CEACAM1, CD66a) is a member of the CEA gene family. CEACAM1 interacts homophilically and heterophilically with CEACAM5, and is involved in various anti-proliferative activities. CEACAM1 is expressed on a variety of epithelial and hematological cells, including multiple types of cancer and activated lymphocytes. High CEACAM1 expression in some tumor types is known to be associated with poor disease prognosis. Recently it was demonstrated CEACAM1 is co-expressed on exhausted lymphocytes with other immune checkpoints such as TIM-3 and may regulate downstream activity. CM24 is a novel humanized α-CEACAM1-specific antibody with nM affinity to the N terminal domain of CEACAM1, which blocks intercellular CEACAM1 interactions. Methods: The primary objective was to test the safety and tolerability of CM24 in adult patients with advanced or recurrent cancer. Secondary objectives included assessment of CM24 PK and PD profiles, anti-tumor response and the recommended Phase 2 dose. Patient received IV infusion of CM24 at 7 dose levels ranging between 0.01 and 10 mg/kg in a cycle of 4 doses administered q2wks followed by a 6-week observation only period and additional 6 cycles. Results: 27 patients (median pretreatment of 4 prior regimens; range 2-8, 11 colorectal, 7 melanoma, 4 ovarian, 3 gastric, 2 NSCLC; 13 males, 14 females, mean age of 60 years), were included. Treatment with CM-24 was overall well-tolerated without DLTs up to 10 mg/kg. The most frequent AE was grade 1-3 increased alanine aminotransferase (7 subjects) and the most severe AE was grade 3/4 increase in gamma-glutamyltransferase (4 subjects). Drug-related AEs were observed in 63% of the subjects with grade 3-5 occurred in 3.7%. Eight subjects (29.6%) had stable disease as the best overall response. Median overall survival was 4 (3.4, 8.0) and 6.2 (2.7, 10.2) months for the 3 and 10 mg/kg doses, suggesting dose response. Cmax, AUC and t1/2 increased with increasing dose with the longest t1/2 of 11.2 days obtained at 10mg/kg. The average target occupancy of CM24 at 3mg/kg and 10mg/kg were 75% and 93%, respectively. Conclusions: PK and target-mediated drug disposition analysis suggest that doses higher than 10mg/kg are needed for target saturation at a q2 week regimen while a q3 week regimen is less optimal. A phase 1/2 clinical trial testing CM24 in combination with anti-PD-1 therapy in patients with NSCLC including assessment of CEACAM1 expression is warranted. Clinical trial information: NCT02346955 .

Natural selection supports escape from concerted evolution of a recently duplicated CEACAM1 paralog in the ruminant CEA gene family

Concerted evolution is often observed in multigene families such as the CEA gene family. As a result, sequence similarity of paralogous genes is significantly higher than expected from their evolutionary distance. Gene conversion, a “copy paste” DNA repair mechanism that transfers sequences from one gene to another and homologous recombination are drivers of concerted evolution. Nevertheless, some gene family members escape concerted evolution and acquire sufficient sequence differences that orthologous genes can be assigned in descendant species. Reasons why some gene family members can escape while others are captured by concerted evolution are poorly understood. By analyzing the entire CEA gene family in cattle (Bos taurus) we identified a member (CEACAM32) that was created by gene duplication and cooption of a unique transmembrane domain exon in the most recent ancestor of ruminants. CEACAM32 shows a unique, testis-specific expression pattern. Phylogenetic analysis indicated that CEACAM32 is not involved in concerted evolution of CEACAM1 paralogs in ruminants. However, analysis of gene conversion events revealed that CEACAM32 is subject to gene conversion but remarkably, these events are found in the leader exon and intron sequences but not in exons coding for the Ig-like domains. These findings suggest that natural selection hinders gene conversion affecting protein sequences of the mature protein and thereby support escape of CEACAM32 from concerted evolution.

Gene expression noise in a complex artificial toxin expression system

Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system.

Six-gene Assay as a new biomarker in the blood of patients with colorectal cancer: establishment and clinical validation.

Colorectal cancer (CRC) is the second most common cancer in men and the third most common cancer in women. Although long‐term survival has improved over the past 30 years, at least 50% of patients with CRC will develop metastases after diagnosis. In this study, we examined whether quantifying the mRNA of six CRC‐related genes in the blood could improve disease assessment through detection of circulating tumor cells (CTC), and thereby improve progression prediction in relapsed CRC patients. Cell spiking assay and RT‐PCR were performed with blood samples from healthy volunteers spiked with six CRC cell lines to generate an algorithm, herein called the Six‐gene Assay, based on six genes (CEA, EpCAM, CK19,MUC1,EGFR and C‐Met) for CTC detection. The CTCs of 50 relapsed CRC patients were then respectively measured by CEA Gene Assay (single‐gene assay control) and Six‐gene Assay. Subsequently, receiver operating characteristic analysis of the CTC panel performance in diagnosing CRC was conducted for both assays. Moreover, the 2‐year progression‐free survival (PFS) of all patients was collected, and the application of CEA Gene Assay and Six‐gene Assay in predicting PFS was carefully evaluated with different CTC cutoff values. Encouragingly, we successfully constructed the first multiple gene‐based algorithm, named the Six‐gene Assay, for CTC detection in CRC patients. Six‐gene Assay was more sensitive than CEA Gene Assay; for instance, in 50 CRC patients, the positive rate of Six‐gene Assay in CTC detection was 82%, whereas that of CEA Gene Assay was only 70%. Moreover, Six‐gene Assay was more sensitive and accurate than CEA Gene Assay in diagnosing CRC as well as predicting the 2‐year PFS of CRC patients. Statistical analysis demonstrated that CTC numbers measured by Six‐gene Assay were significantly associated with 2‐year PFS. This novel Six‐gene Assay improves the definition of disease status and correlates with PFS in relapsed CRC, and thus holds promise for future clinical applications.

Alternative splicing after gene duplication drives CEACAM1-paralog diversification in the horse

BackgroundThe CEA gene family is one of the most rapidly evolving gene families in the human genome. The founder gene of the family is thought to be an ancestor of the inhibitory immune checkpoint molecule CEACAM1. Comprehensive analyses of mammalian genomes showed that the CEA gene family is subject to tremendous gene family expansion and contraction events in different mammalian species. While in some species (e.g. rabbits) less than three CEACAM1 related genes exist, were in others (certain microbat species) up to 100 CEACAM1 paralogs identified. We have recently reported that the horse has also an extended CEA gene family. Since mechanisms of gene family expansion and diversification are not well understood we aimed to analyze the equine CEA gene family in detail.ResultsWe found that the equine CEA gene family contains 17 functional CEACAM1-related genes. Nine of them were secreted molecules and eight CEACAMs contain transmembrane and cytoplasmic domain exons, the latter being in the focus of the present report. Only one (CEACAM41) gene has exons coding for activating signaling motifs all other CEACAM1 paralogs contain cytoplasmic exons similar to that of the inhibitory receptor CEACAM1. However, cloning of cDNAs showed that only one CEACAM1 paralog contain functional immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Three receptors have acquired a stop codon in the transmembrane domain and two have lost their inhibitory motifs due to alternative splicing events. In addition, alternative splicing eliminated the transmembrane exon sequence of the putative activating receptor, rendering it to a secreted molecule. Transfection of eukaryotic cells with FLAG-tagged alternatively spliced CEACAMs indicates that they can be expressed in vivo. Thus detection of CEACAM41 mRNA in activated PBMC suggests that CEACAM41 is secreted by lymphoid cells upon activation.ConclusionsThe results of our study demonstrate that alternative splicing after gene duplication is a potent mechanism to accelerate functional diversification of the equine CEA gene family members. This potent mechanism has created novel CEACAM receptors with unique signaling capacities and secreted CEACAMs which potentially enables equine lymphoid cells to control distantly located immune cells.

Expression of Colocasia esculenta tuber agglutinin in Indian mustard provides resistance against Lipaphis erysimi and the expressed protein is non-allergenic.

Transgenic Brassica juncea plants expressing Colocasia esculenta tuber agglutinin (CEA) shows the non-allergenic nature of the expressed protein leading to enhanced mortality and reduced fecundity of mustard aphid-Lipaphis erysimi. Lipaphis erysimi (common name: mustard aphid) is the most devastating sucking insect pest of Indian mustard (Brassica juncea L.). Colocasia esculenta tuber agglutinin (CEA), a GNA (Galanthus nivalis agglutinin)-related lectin has previously been reported by the present group to be effective against a wide array of hemipteran insects in artificial diet-based bioassays. In the present study, efficacy of CEA in controlling L. erysimi has been established through the development of transgenic B. juncea expressing this novel lectin. Southern hybridization of the transgenic plants confirmed stable integration of cea gene. Expression of CEA in T0, T1 and T2 transgenic plants was confirmed through western blot analysis. Level of expression of CEA in the T2 transgenic B. juncea ranged from 0.2 to 0.47% of the total soluble protein. In the in planta insect bioassays, the CEA expressing B. juncea lines exhibited enhanced insect mortality of 70-81.67%, whereas fecundity of L. erysimi was reduced by 49.35-62.11% compared to the control plants. Biosafety assessment of the transgenic B. juncea protein containing CEA was carried out by weight of evidence approach following the recommendations by FAO/WHO (Evaluation of the allergenicity of genetically modified foods: report of a joint FAO/WHO expert consultation, 22-25 Jan, Rome, http://www.fao.org/docrep/007/y0820e/y0820e00.HTM , 2001), Codex (Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome; Codex, Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome, 2003) and ICMR (Indian Council of Medical Research, guidelines for safety assessment of food derived from genetically engineered plants, http://www.icmr.nic.in/guide/Guidelines%20for%20Genetically%20Engineered%20Plants.pdf , 2008). Bioinformatics analysis, pepsin digestibility, thermal stability assay, immuno-screening and allergenicity assessment in BALB/c mice model demonstrated that the expressed CEA protein from transgenic B. juncea does not incite any allergenic response. The present study establishes CEA as an efficient insecticidal and non-allergenic protein to be utilized for controlling mustard aphid and similar hemipteran insects through the development of genetically modified plants.

CEA-EPO质粒的构建及诱导造血干细胞定向生成红细胞疫苗抗结肠癌的研究

目的：研究CEA-EPO质粒的构建及诱导造血干细胞定向生成红细胞疫苗抗结肠癌的研究。方法：将EPO-cDNA插入pcDNA3.1质粒的多克隆位点，用CEA基因启动子替代pcDNA3.1质粒的CMV启动子，构建成CEA-EPO重组质粒。用含抗体CD117磁珠分离纯化和收集小鼠骨髓造血干细胞，用重组腺病毒法将CEA-EPO基因体外转染CEA-EPOmRNA基因到造血干细胞内，检测CEA-EPO基因和蛋白表达，将疫苗注入小鼠体内，进行扩增，使造血干细胞定向生成携带CEA-EPO双基因红细胞，经过提取和纯化获得CEA-EPO基因共转染红细胞疫苗，再进行双基因共转染红细胞疫苗的体外和体内实验。结果：成功收集造血干细胞，流式细胞仪分析结果表明：纯化前后有明显差异(P < 0.05)；检测CEA-EPOmRNA和蛋白表达的结果表明：造血干细胞体外转染CEA-EPOmRNA基因成功，获得CEA-EPO双基因共转染红细胞疫苗；应用细胞红系相关标志抗体GPA和CD71标记，利用流式细胞证实红细胞疫苗培养成功；研究相关疫苗激活的单核细胞对结肠癌细胞株CT26细胞杀伤作用表明：在比例为40:1时，CEA-EPO疫苗组杀伤作用为57.34 ± 1.13，高于空载组、CEA疫苗组和EPO疫苗组(P < 0.05)，体内试验的成瘤性研究表明：CEA-EPO疫苗组肿瘤的生长的大小为0.29 ± 0.11 (cm3)；高于其他各组的大小(P < 0.05)，CEA-EPO疫苗组的成活期分别为：69 ± 6.91天，高于其他各组的大小(P < 0.05)；CEA-EPO疫苗组在肿瘤大小和小鼠的生存时间上，与空载组、CEA疫苗组，EPO疫苗组之间差异有统计学意义(P < 0.05)。结论：携带CEA-EPO双基因肿瘤抗原的红细胞疫苗对结肠癌有明显的抗肿瘤作用，既可以诱导造血干细胞定向分化成红细胞疫苗，又可以合成肿瘤抗原的红细胞疫苗，是临床治疗结肠的重要疫苗。 Objective: To investigate the effects of recombinant adenovirus-mediated co-transfection of CEA gene and EPO gene on promoting hematopoietic stem cell directly producing erythrocyte vaccine against colon cancer. Method: The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus car-rying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived MSCs of mice were isolated and cultured in vitro by anti-CD117magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP(GFP group), co-transfected with CEA and EPO (CEA and EPO group), or transfected with no virus (controlgroup). The expression of CEA and EPO gene and protein after transfection in supernatant fluid of culture was detected by RT-PCR and western blot in each group. We have checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. Result: We have successfully gathered the hematopoietic stem cells; flow cytometry analysis result showed that there were significant differences before and after purification (P < 0.05). The expression of double genes (CEA-EPO gene) and protein shows CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We have confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that CEA-EPO group was 57.34 ± 1.13% in proportion of 40:1. Compared with CEAg group and EPO group, there was significant difference between them (P < 0.05). In the experimentation of neoplasma format, the size of murine growth of tumor was 0.29 ± 0.11 cm3 differently in the group of CEA-EPO group; the number of murine live time on day was 69 ± 6.91 days. Compared with the group of CEA and plasmid, EPO, the group of CEA-EPO was significant differences in size and live time (P < 0.05). Conclusion: The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. It not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor Antigen vaccine against stomach cancer.

Two Molecular Markers of Early Non-Small Cell Lung Carcinoma Based on Gene Expression in Peripheral Blood

Background: Lung cancer is among the most common cancers. Search is ongoing to find biomarkers to improve the diagnosis lung cancer techniques in early stages. In this study we evaluate the sensitivity and specificity of the MUC1 and CEA gene expressions in the peripheral blood of non-small cell lung cancer (NSCLC). Material and Methods: This study was done in Masih Daneshvari Hospital, Tehran, Iran and was case/control study that conducted on 30 NSCLC patients and 30 healthy controls. Peripheral blood was collected and total RNA was extracted then cDNA was synthesized. Sample was separately assessed by real time PCR. Results: The expression of CEA gen was positive in 24 patients indicating 80% sensitivity for this marker. The expression of CEA gen was positive in 9 controls out of 30 each. A statistically significant difference was detected between patients and healthy controls with regard to CEA mRNA expression (P < 0.001). The MUC1 gen expressed in 20 out of 30 patients, while it expressed in 3 controls. The difference in MUC1 mRNA expression was statistically significant between NSCLC patients and healthy controls (P < 0.001). Conclusion: MUC1 and CEA are molecular biomarkers with relatively favorable sensitivity for primary diagnosis of NSCLC.

Hierarchical Post-transcriptional Regulation of Colicin E2 Expression in Escherichia coli.

Post-transcriptional regulation of gene expression plays a crucial role in many bacterial pathways. In particular, the translation of mRNA can be regulated by trans-acting, small, non-coding RNAs (sRNAs) or mRNA-binding proteins, each of which has been successfully treated theoretically using two-component models. An important system that includes a combination of these modes of post-transcriptional regulation is the Colicin E2 system. DNA damage, by triggering the SOS response, leads to the heterogeneous expression of the Colicin E2 operon including the cea gene encoding the toxin colicin E2, and the cel gene that codes for the induction of cell lysis and release of colicin. Although previous studies have uncovered the system’s basic regulatory interactions, its dynamical behavior is still unknown. Here, we develop a simple, yet comprehensive, mathematical model of the colicin E2 regulatory network, and study its dynamics. Its post-transcriptional regulation can be reduced to three hierarchically ordered components: the mRNA including the cel gene, the mRNA-binding protein CsrA, and an effective sRNA that regulates CsrA. We demonstrate that the stationary state of this system exhibits a pronounced threshold in the abundance of free mRNA. As post-transcriptional regulation is known to be noisy, we performed a detailed stochastic analysis, and found fluctuations to be largest at production rates close to the threshold. The magnitude of fluctuations can be tuned by the rate of production of the sRNA. To study the dynamics in response to an SOS signal, we incorporated the LexA-RecA SOS response network into our model. We found that CsrA regulation filtered out short-lived activation peaks and caused a delay in lysis gene expression for prolonged SOS signals, which is also seen in experiments. Moreover, we showed that a stochastic SOS signal creates a broad lysis time distribution. Our model thus theoretically describes Colicin E2 expression dynamics in detail and reveals the importance of the specific regulatory components for the timing of toxin release.

The cancer vaccine therapy using DCs derived from iPS cells (iPSDCs) expressing TAA gene.

768 Background: Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play a critical role in the initiation of anti-tumor immune responses. Many cancer patients have previously been treated by the cancer vaccine therapy using DCs worldwide. We have employed a study of a cancer vaccine therapy using genetically modified DCs expressing tumor-associated antigen (TAA) gene. Clinically DCs are generated from the peripheral blood monocytes of patients. Thus the number of monocytes and potential of them are limited, so they are serious obstacle. Recent studies have revealed that induced pluripotent stem (iPS) cells can be generated from murine fibroblasts. Furthermore, it has been reported that DCs can be successfully derived from murine iPS cells (iPSDCs). If the therapeutic efficacy of iPSDCs is equivalent to that of naive DCs, then the above-mentioned problems may be solved. Methods: We have induced iPSDCs from murine iPS cells by 4 steps and examined the efficacy as APCs of iPSDCs compared with naive DCs. We also examined whether a vaccine therapy using genetically modified iPSDCs can induce strong therapeutic antitumor immunity compared with naive DCs. Next, we examined the therapeutic antitumor immunity of iPSDCs expressing CEA gene compared with that of naive DCs in pre-clinical study with CEA transgenic mice. Results: We have clarified that genetically modified iPSDCs have an equal efficacy as APCs and TAA-specific therapeutic antitumor immunity, equivalent to naive DCs. And we also have clarified that genetically modified iPSDCs expressing CEA gene have a TAA-specific therapeutic antitumor immunity. Conclusions: This vaccine strategy using genetically modified iPSDCs has an equal capacity with naive DCs in terms of a therapeutic efficacy. Now, we are engaging another pre-clinical study with human, in an effort to apply in a clinical setting.

Amount of colicin release in Escherichia coli is regulated by lysis gene expression of the colicin E2 operon.

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased.

Expression of MAGE genes peripheral blood of gastric cancer patients and its relation to lymph node metastasis and prognosis

Objective In the detection of peripheral blood in patients with gastric cancer the melanoma antigen A1 (MAGEA1) and the melanoma antigen A3 (MAGEA3) gene expression,combined with the conditions of patients with postoperative follow-up analysis MAGE gene expression and the relationship between gastric cancer metastasis and prognosis.Methods Peripheral blood was obtained from 40 patients with gastric carcinoma and 20 patients with benign diseases.The mRNA of the MAGEA1 and MAGEA3 genes in the peripheral blood mononuclear cells(PBMC) was detected by RT-PCR,meanwhile the MAGEA1 and MAGEA3 transcripts in gastric carcinoma tissues and corresponding nomal tissue were detected.In contrast with CEA gene,we detected its expression by nested RT-PCR.Results Of the 40 gastric carcinoma patients,MAGEA1 and MAGEA3 mRNA were positive in 47.5％ (17/40) and 25.0％ (10/40) of PBMC,respectively,and in 65.0％ (26/40) and 30.0％ (12/40) of gastric carcinoma tissues,the corresponding paracancerous gastric tissues were not expressed,of which 4 cases of peripheral blood both expression.In the PBMC of 40 gastric carcinoma patients,62.5％ (25/40) samples were detected to express at least one type of MAGE mRNA,the detection of CEA gene mRNA in peripheral blood of the patients in PBMCwas 65.0％ (26/40),20 patients with non neoplastic diseases of PBMC were not detected in the MAGE gene mRNA.There were 22.5％ (9/40) of 40 gastric carcinoma patients with CEA gene expression in PBMC.The detection rate of the MAGE gene of mRNA PBMC in patients with gastric cancer was not related with the lymph node metastasis,TNM stage,and the degree of tumor differentiation,depth of invasion,tumor size,distant metastasis.Conclusion Gastric cancer patients with peripheral blood MAGE gene expression is closely related to the tumor lymph node metastasis,TNM staging,express positive patients with poor prognosis. Key words: Gastric neoplasms ; Melanoma antigen gene ; Peripheral blood mononuclear cells; Prognosis

Effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma

To study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity. Stable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo. The cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05). The results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.

Detection of tumor cells in peripheral blood of patients with gastric cancer using mRNA of MAGE genes as markers

To develop a sensitive and specific RT-PCR assay using the mRNA of MAGE-1 and MAGE-3 genes as specific tumor markers for the detection of the tumor cells in the peripheral blood of patients with gastric cancer. Peripheral blood was obtained from 40 patients with gastric cancer and from 20 healthy volunteers. The mRNA of MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMC) was detected by RT-PCR. The expressions of MAGE-1 and MAGE-3 mRNA in the tumor tissues of these gastric cancer patients were also detected by RT-PCR. Meanwhile,CEA expression by nested RT-PCR in PBMC of 40 gastric cancer patients was also detected. Of 40 gastric cancer patients, MAGE-1 and MAGE-3 mRNA were positive in 47.5% (19/40) and 25% (10/40) of PBMC respectively, and in 62.5% (25/40) and 30% (12/40) of gastric cancer tissues respectively. As a whole, in the PBMC of 40 gastric cancer patients, 25 (62.5%) samples were found to express at least one type of MAGE mRNA. In the patients whose tumors did not express MAGE-1 and/or MAGE-3 genes, the corresponding MAGE mRNA was also undetected in their PBMC. There was no expression of MAGE-1 or MAGE-3 gene in the PBMC from the 20 healthy donors. The positive rate of MAGE mRNA in PBMC was closely correlated with the tumor stage and lymph node metastasis (P <0.05). Positive rate of CEA gene expression was 32.5% (13/40) in the PBMC of 40 gastric cancer patients, 29 (72.5%)samples were detected to express at least one type of MAGE gene and CEA gene mRNA. MAGE-1, MAGE-3 and CEA mRNA are specifically detected with high percentage in the PBMC of gastric cancer patients by RT-PCR. They could be used as specific tumor markers for the detection of the circulating gastric cancer cells, and the detection results may be helpful to evaluate the prognosis of gastric cancer patients.

A new way for isolation and cultivation of sweat gland ductal cells from human split-thickness skin in vitro

To explore a new method of isolation and culture of eccrine sweat gland ductal cells from human split-thickness skin graft in vitro. Human split-thickness skin graft which was presented by volunteer (n = 10) was digested with type II collagenase, and then sweat gland duct were isolated from the split-thickness skin graft, primary cultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2, 95% O2. The cultured eccrine sweat gland ductal cells were identified by analysis CEA, CK8, CK18, CK19 antigens expression with flow cytometry, RT-PCR and Western Blot, and by detecting the electrophysiology with whole cell patch clamp technology. The isolated eccrine sweat gland ductal cells could grow by adhering to the wall, proliferate in vitro after 48 h of adhering to the wall, and confluens after 2 - 4 weeks of adhering to the wall. The FACs analysis showed the expression of CEA was (90.26 +/- 1.12)%, (89.70 +/- 1.43)%, and CK8 was (94.41 +/- 1.84)%, (93.65 +/- 1.63)% in primary cultured sweat gland ductal cells and primary cultured eccrine sweat gland cells, respectively, and there is no significant difference between the two groups (P > 0.05). Immunocytochemistry staining showed CEA, CK8, CK18, CK19 was positive in sweat gland duct cells, RT-PCR revealed that CEA, CK8, CK18 and CK19 gene expression in sweat gland ductal cells, and Western Blot analysis showed the expression of CEA brand, CK8 brand, CK18 brand, and CK19 brand in sweat gland ductal cells, patch clamp indicated that this cells has distinct amiloride sensitive Na(+) channels. The cultured human eccrine sweat gland duct cells in vitro display the markers and biological characteristics of sweat gland epithelial lineage, and this method of digest the split-thickness skin graft to get the sweat gland duct cells is better than classical dissect sweat gland under dissect microscope.

Expression and clinical significance of CEA-mRNA, CK20-mRNA in peripheral blood of esophageal cancer patients

Objective To establish reverse transcriptase-polymerase chain reaction(RT-PCR) with primers specific for CEA gene and CK20 gene to detect circulating tumor cells in peripheral blood of esophageal cancer pa-tients,and try to find the relationship between the mRNA expression and micrometastasis. Methods The expressions of CEA,CK20 were analyzed by RT-PCR in 53 cases of esophageal tumor tissue and in peripheral blood,compared with 10 patients with benign esophageal disease and 20 healthy volunteers. Results The expressions of CEA-mRNA, CK20-mRNA were 96.23% (51/53), 100% ( 53/53 ) in 53 esophageal tumor tissue and were 52.83% (28/53), 49.06% (26/53) in peripheral blood of 53 esophageal cancer patients. In control group,there was only one expression of CEA-mRNA in peripheral blood of 10 patients with benign esophageal disease,as well as in 20 healthy volunteers. There was no expression of CK20-mRNA in peripheral blood of 10 patients with benign esophageal disease and 20 healthy volunteers. Conclusion CEA-mRNA, CK20-mRNA might be specific and sensitive markers to detect circu-lating tumor cells in peripheral blood and their expression was closely related to TNM stages of the esophageal cancer patients. Key words: Esophageal neoplasms; Neoplasm metastasis; Carcinoembryonic antigen; Keratin-20

Generation of paired immune receptors by gene conversion within the canine CEA gene family

Generation of paired immune receptors by gene conversion within the canine CEA gene family