Retraction Note: HAX-1 Promotes the Chemoresistance, Invasion, and Tumorigenicity of Esophageal Squamous Carcinoma Cells.

The Editor-in-Chief has retracted this article [1] because Figure 3c appears to have been modified and reused as Figure 3d.

Down-regulation of PTEN expression modulated by dysregulated miR-21 contributes to the progression of esophageal cancer.

BACKGROUND AND AIM: miR-21, a putative tumor oncomiR, is a frequently overexpressed miRNA in a variety of tumors. Because it targets tumor-suppressor genes it has been linked to tumor progression. In this study we investigated the role of miR-21 in esophageal squamous cell carcinoma (ESCC), and its possible mechanism. METHODS: Expression of miR-21 was detected by stem-loop RT-PCR in tissue from 76 invasive ESCC at stage I-IV and in their corresponding para-cancerous histological normal tissues (PCHNT). Thirty endoscopic esophageal mucosal biopsy specimens from non-tumor patients were used as controls. Expression of PTEN in 76 paired ESCC and PCHNT was investigated by real-time RT-PCR and an immunohistochemical method, respectively. Paired tumor and PCHNT specimens of 20 ESCC cases were randomly selected for western blot analysis. The effect of miR-21 on PTEN expression was assessed in the ESCC cell line with an miR-21 inhibitor to reduce miR-21 expression. Furthermore, the roles of miR-21 in cell biology were analyzed by use of miR-21 inhibitor-transfected cells. RESULTS: Stem-loop RT-PCR revealed miR-21 was significantly overexpressed in ESCC tissues and cell lines. Overexpression of miR-21 correlated with tumor status, lymph node metastasis, and clinical stage. We demonstrated that knockdown of miR-21 significantly increased expression of PTEN protein. Consequent PTEN expression reduced cell proliferation, invasion, and migration. CONCLUSIONS: Our findings suggest that miR-21 could be a potential oncomiR, probably by regulation of PTEN, and a novel prognostic factor for ESCC patients.

Epigenetic inactivation of Wnt inhibitory factor-1 in human esophageal squamous cell carcinoma.

Wnt inhibitory factor-1 (WIF1), as one of most important Wnt antagonists, has been detected frequently silenced by promoter hypermethylation in various types of cancer. In this study, we aimed to investigate the promoter methylation profiles of WIF1 in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines, as well as the functional roles of WIF1 in the human ESCC metastatic behavior. WIF1 mRNA levels and promoter methylation status in ESCC tissues and cell lines were detected using RT-PCR and methylation-specific PCR (MS-PCR), respectively. WIF1 protein levels were assessed by Western blot. Stable ESCC cell line with restoration of WIF1 was generated in EC109 cells, which naturally do not express detectable WIF1 mRNA. The effects of reexpressed WIF1 on EC109 cell proliferation and migration were investigated using crystal violet and wound healing assay, respectively. Also the effects of WIF1 reexpression on the beta-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay. WIF1 promoter methylation was frequently observed in ESCC tissues (46%, 23/50) and cell lines (50%, 2/4). Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), increased or restored WIF1 expression in these ESCC cell lines. Restoration of the WIF1 in EC109 cells resulted in a significant inhibition on both cell proliferation and migration. Moreover, reexpression of WIF1 caused significant decrease of beta-catenin/T-cell factor-dependent transcription activity. These findings demonstrated that WIF1 silencing due to promoter hypermethylation is a major mechanism during carcinogenesis of ESCC. This would be an opportunity to prevent the development and progression of HCC through modulation of WIF1.

HAX-1 promotes the chemoresistance, invasion, and tumorigenicity of esophageal squamous carcinoma cells.

BACKGROUND: HAX-1 is an anti-apoptotic factor and regulates the expression of DNA pol ß. Interestingly, DNA polymerase pol ß is overexpressed in esophageal squamous cell carcinoma (ESCC). However, the functional role of HAX-1 in ESCC remains unclear. AIMS: To investigate the role of HAX-1 in chemoresistance, invasion, and tumorigenicity of ESCC. METHODS: Lentivirus-mediated overexpression or knockdown of HAX-1 was employed to establish ESCC EC9706 cell lines that expressed HAX-1 at different levels. The biological behaviors of these engineered cells were characterized in vitro and in vivo using a xenograft nude mice model. In addition, HAX-1 and pol ß expression in the tumor tissues was detected by RT-PCR and immunohistochemistry. RESULTS: HAX-1 overexpression promoted cell proliferation and resistance against cisplatin, increased cell invasion and suppressed apoptosis along with increased pol ß expression. Conversely, HAX-1 knockdown inhibited the malignant phenotypes of EC9706 cells. The xenograft nude mice model demonstrated that HAX-1 overexpression or depletion led to increased or decreased tumor growth in vivo, respectively. Furthermore, a positive correlation of HAX-1 and pol ß expression in the tumor tissues was observed. CONCLUSIONS: HAX-1 promotes the proliferation, chemoresistance, invasion, and tumorigenicity of ESCC, and this is correlated with increased poly ß expression. HAX-1 may represent a potential target to overcome the resistance and metastasis of ESCC.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Study on the specific immunity induced by dendritic cell vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell in mice].

OBJECTIVE: To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice. METHODS: Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R2) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD3+CD8+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. RESULTS: The expression of VEGF-R2 and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD3+CD8+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R2). CONCLUSIONS: bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.

[Expression and prognostic significance of EphA2 and EphrinA-1 in ovarian serous carcinomas].

OBJECTIVE: To investigate the expression and prognostic significance of EphA2 and EphrinA-1 in ovarian serous carcinomas. METHODS: Ninety five tumors from the patients with ovarian serous carcinomas and 2 ovarian cancer cell lines were recruited. The expressions of EphA2 and EphrinA-1 were examined by means of immunohistochemistry. The relationships among protein expression and clinicopathological features, survival of patients were analyzed. The mRNA and protein expressions of EphA2 and EphrinA-1 in ovarian cancer cell lines were measured with semiquantitative polymerase chain reaction and western blotting. RESULTS: The protein expressions of EphA2 and EphrinA-1 in tumor were 92.6% (88/95)and 97.9% (93/95) respectively, while were 40%(8/20) and 30% (6/20) in adjacent ovarian tissue (P = 0.000). EphA2 immunohistochemical staining could be observed in both tumour and vascular endothelial cells, and EphrinA-1 mainly localized in the tumor cells. The expression of EphA2 was significantly associated with the expression of EphrinA-1 (r = 0.98, P = 0.02). There was no significant correlation between the expressions of EphA2/EphrinA-1 and age, FIGO stage, residual tumour size and histological grade. High levels of both EphA2 and EphrinA-1 protein expression were significantly associated with a shorter overall survival in multivariate analysis (P < 0.05). High levels of EphA2 and EphrinA-1 mRNA and protein were detected in OVCAR3 and SKOV3 cell lines. CONCLUSION: There are over-expression of EphA2 and EphrinA-1 in ovarian serous carcinomas and ovarian cancer cell lines. Tumours with higher expression levels of both EphA2 and EphrinA-1 significantly associated with poorer clinical outcome.

[Preliminary study of DNA polymerase beta gene silencing by small interfering RNA in human gastric cancer BGC-823 cells].

OBJECTIVE: To study the influence of DNA polymerase beta (polbeta) gene silencing by small interfering RNA on biological behavior of human gastric cancer cell line BGC-823. METHODS: The siRNA eukaryotic expression vectors targeting polbeta gene were constructed and transfected into BGC-823 cells by liposome. Stable cell lines were screened with G418. The expression levels of polbeta mRNA and protein were detected by real time PCR and Western blot in the cells of each group. The proliferation of each group was detected by flow cytometry and tumorigenicity was determined in nude mice. RESULTS: The siRNA expression vector targeting polbeta gene was successfully constructed. The expression levels of polbeta mRNA and protein were significantly reduced in the experimental group transfected with siRNA expression vectors targeting polbeta, and the silencing effect of pRNAT-U6.1-sipolbeta2 (suppression degree was 83%) was stronger than that of pRNAT-U6.1-sipolbeta1 (depression degree is 56%). Compared with irrelevant siRNA control group, empty vector control group and untransfected group, the ratio of G0/G1 cells was increased, proportion of S phase cells and cell proliferation were decreased in the experimental group 1 cells transfected with pRNAT-U6.1-sipolbeta1 (P < 0.05). On the contrary, the ratio of G1/G0 was decreased, proportion of S phase cells and cell proliferation was increased in the experimental group 2 cells transfected with pRNAT-U6.1-sipolbeta2 (P < 0.05). CONCLUSION: The siRNA expression vectors targeting DNA polymerase beta gene can significantly inhibit the expression of polbeta mRNA. Neither high nor extremely low expression of polbeta is beneficial to maintain the cellular physiological functions. The expression of polbeta silenced to a proper level by siRNA may play an important role in inhibiting tumorigenesis.

Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450.

In current study, we investigated the anti-tumor effect of luteolin in human ESCC cell lines in vitro and in vivo and tried to explore the potential mechanisms. Results from flow cytometry showed that luteolin could induce apoptosis and caspase-3 activation and induce cell cycle arrest at G2/M phase in a dose- and time-dependent manner in EC1 and KYSE450 cells. JC-1 test results showed that membrane potential of mitochondria after luteolin treatment was down-regulated and this was an indicator for intrinsic apoptosis. Western Blot results showed the expression of cell cycle regulatory protein p21 and p53 increased and three apoptosis related proteins that participate in mitochondrial apoptotic pathway, namely, Bim, CYT-c and cPARP, also increased in luteolin treated cells compared with control groups. We further confirmed that luteolin could significantly inhibit the growth of ESCC tumors in xenograft mouse models and no evidence of systemic toxicity was observed. Our results suggest that luteolin can induce cell apoptosis and cell cycle arrest in G2/M phase through mitochondrial pathway in EC1 and KYSE450 cell lines and proper utilization of luteolin might be a practical approach in ESCC chemotherapy.

[Study on the association of polymorphisms in homocysteine metabolism related enzymes with deep venous thrombosis].

OBJECTIVE: To explore the significance of gene mutation of methylenetetrahydrofolate reductase (MTHFR) C677T, methionine synthase (MS) 2756 AG and cystathionine beta-synthase (CBS) 844ins68 in the development of deep venous thrombosis. METHODS: One hundred and three cases of deep venous thrombosis (DVT group) and 250 healthy subjects (control group) were recruited in the study. The polymorphisms of MTHFR C677T, MS A2756G and CBS 844ins68 were detected by PCR-restriction fragment length polymorphism(PCR-RFLP). RESULTS: The prevalences of TT genotypes of MTHFR (C677T) between DVT group and normal control group had significant difference (27.2% vs 17.2%, P< 0.05), the prevalence of AG genotypes of MS A2756G in the DVT group was less than that in the control group (9.7% vs 19.2%, P< 0.05). The prevalence of 677T-2756A haplotype in the DVT group was higher than that in the control group (P< 0.05), the prevalence of 677C-2756A haplotype in the DVT group was less than that in the control group (P< 0.05). There were no significant differences in the prevalences of CBS 844ins68 mutation. CONCLUSION: The homozygote of MTHFR C677T (TT) may be a risk factor of DVT. MS A2756 G(AG) genotypes may reduce the development of DVT. The 677T-2756A haplotype may be a risk factor of DVT. The 677C-2756A haplotype may be a protective factor of DVT. The prevalence of gene mutation of CBS 844ins68 might vary with different ethnic group or geographic regions.

[Expressions of estrogen receptor subtypes in epithelial ovarian carcinomas].

OBJECTIVE: To investigate the expressions of estrogen receptor(ER) subtypes ERa and ERP in epithelial ovarian carcinomas. METHODS: One hundred and eighteen Norwegian patients with epithelial ovarian carcinoma were included in this study. The expressions of ERalpha and ERbeta were examined by means of immunohistochemistry. The relationships between protein expressions and clinicopathological features and survival were analyzed. Frozen tissues from 10 cases in which the tumors showed variable ERalpha and ERbeta protein expressions were used for Laser capture microdissection (LCM). Cancer cells in each frozen section were captured with the LCM method and processed for Western blot analysis. RESULTS: Of the 118 tumours, 32 (27.1%), 20 (16.9%), 17 (14.4%), 49 (41.5%) demonstrated negative, weak, moderate and strong expression of ERalpha protein, respectively; 1 (0.8%), 7 (5.9%), 13 (11%), 97 (82.2%) showed negative, weak, moderate and strong expressions of ERbeta protein, respectively. There was no significant association between ERalpha expression and the clinicopathological features such as age, histological type, FIGO stage, histological grade and residual tumour size. ERbeta expression was not associated with age, histological type,FIGO stage and residual tumour size, but it was significantly associated with higher histological grade. In addition, Kaplan-meier analysis revealed that high levels of ERbeta expression was significantly associated with a shorter overall survival (P = 0.03). CONCLUSION: There are ERalpha and ERbeta expressions in epithelial ovarian carcinomas. Higher level of ERbeta protein expression is associated with higher histological grade and poorer clinical outcome in the cases of ovarian carcinoma. ERbeta may be a useful marker of ovarian carcinogenesis and could predict the efficacy of endocrinotherapy and the prognosis for patients with ovarian carcinoma.

Mutation of DNA polymerase beta in esophageal carcinoma of different regions.

AIM: To observe the variation of DNA polymerase beta (polbeta) in esophageal carcinoma. METHODS: Thirty specimens containing adjacent normal epithelial tissues were collected from patients in Linzhou region (a high risk area for esophageal squamous carcinoma) and 25 specimens were from a non-high risk area. Total RNA was extracted from the samples and reverse transcription polymerase chain reaction (RT-PCR) was performed. PCR products were cloned and sequenced to investigate the polbeta gene with DNASIS and OMIGA. Statistical significance was evaluated using the chi(2) test. RESULTS: High-incidence area group: polbeta gene variation was detected in 13 of 30 esophageal carcinoma tissue specimens, and only one variation was found in 30 corresponding adjacent normal tissue specimens. Non high-incidence area group: polbeta gene variation was detected in 5 of 25 esophageal carcinoma tissue specimens, and no variation was found in 25 corresponding adjacent normal tissue specimens. The incidence of polbeta gene variation observed in the high-incidence area group was significantly higher than in the non-high incidence area group. Two mutation hot spots (454-466 and 648-670 nt) and a 58 bp deletion (177-234 nt) were found. CONCLUSION: Variations of polbeta perform different functions between the high-incidence areas and the other areas, and may play a more important role in the high-incidence areas.

Epigenetic silencing of HIC1 promotes epithelial-mesenchymal transition and drives progression in esophageal squamous cell carcinoma.

Downregulation of the novel tumor suppressor gene HIC1 (hypermethylated in cancer 1) occurs frequently in various tumors where it causes tumor progression and metastasis. In this study, we investigated a role of HIC1 in esophageal squamous cell carcinoma (ESCC) and the underlying mechanisms. Downregulation of HIC1 occurred in approximately 70% of primary ESCCs at both mRNA and protein level where it was associated significantly with vascular invasion, advanced clinical stage, lymph node metastasis, and poor disease free survival (DFS). The promoter methylation analyses suggested that loss of HIC1 expression was mediated by epigenetic mechanisms. Functional studies established that ectopic re-expression of HIC1 in ESCC cells inhibited cell proliferation, clonogenicity, cell motility, tumor formation and epithelial-mesenchymal transition (EMT). Our results decipher the mechanism through which HIC1 deficiency induce ESCC cells to undergo EMT and promote tumor progression and metastasis through activation of EphA2 signaling pathway. Together, loss of the regulation of EphA2 pathway through HIC1 epigenetic silencing could be an important mechanism in the ESCC progression. We identify a novel pathway that linking HIC1 downregulation to EphA2-inducing EMT in ESCC cells and may shed light on the development of novel anti-tumor therapeutics.

[Study on DNA polymerase beta gene mutation in human cervical cancer].

OBJECTIVE: To investigate whether DNA polymerase beta (POLB) gene mutations occur in human cervical cancer (CC). METHODS: To collect fresh specimens from 34 cervical cancer and examine the mutation of POLB gene using reverse transcription polymerase chain reaction (RT-PCR), single strand conformation polymorphism (SSCP) analysis and sequence analysis. According to their histological grading, the 34 cases of CC were divided into three groups: 9 cases of grade I (G(1)), 14 cases of grade II (G(2)), 11 cases of grade III (G(3)). RESULTS: POLB mutations were detected in the tissues of CC. The mutation of POLB is related with the histological differentiated of the CC. The mutated rates in low differentiated cancer were significantly higher than those in moderate and high ones, and the difference was significant (P < 0.05). The sequencing of the PCR product showed an A to G chang at nucleotide 660, which resulted in a substitution of the amino acid from arginine to glycin at codon 182. CONCLUSIONS: There were POLB mutations in the tissues of CC, and this may be related with the development of the CC. The mutations impaired the catalytic activity of POLB. As a result, this may lead to observed accumulation of mutations in tumor cells.

[Rapid detection of the genotyping of hepatitis C virus using DNA chip with coloration methods].

OBJECTIVE: To develop a new DNA chip with coloration, which can be used for rapid and economical detection of the genotyping of hepatitis C virus (HCV). METHODS: Probes and primers were designed according to the sequence of HCV 5' non-coding region (5' NCR) to fabricate DNA chip. Experimental group consisted of 60 positive serum samples and control group consisted of 20 negative serum samples. To obtain the aimed gene, then they were hybridized with DNA chip. Finally, the results showed in a nylon film. The results of DNA sequencing of samples were used as the control in double blind experimental. RESULTS: Using DNA chip, HCV was detected in positive of all serum specimens of experimental group and negative in control group. The determination of HCV genotype by DNA chip showed corresponding rate of 96.7% with those by sequence assay. CONCLUSION: It showed higher specialty and sensitivity using DNA chip to detect the genotype of HCV. It would be valuable for the clinical genotyping of HCV

Roles of GST-π and polß genes in chemoresistance of esophageal carcinoma cells.

The main aim of this study was to investigate the roles of GST-π and polß genes in the chemoresistance of esophageal carcinoma cells. Eukaryotic expression vectors containing each gene were constructed and transfected into EC9706 cells, and the biological effects of the two genes assessed based on a resistance index. We additionally investigated the in vitro and in vivo anti-resistance effects of GST-π and polß genes using recombinant lentiviruses carrying siRNAs against the two genes. Our results showed that upregulation of GST-π and polß genes suppresses chemosensitivity of esophageal carcinoma cells to cisplatin, while downregulation of these two genes with RNAi technology reverses this chemoresistance. Multi-site injection of recombinant lentivirus targeting the GST-π gene into transplanted cDDP tumors effectively reversed their chemoresistant phenotype. However, the same treatment against the polß gene did not lead to significant efficacy against chemoresistance.

Enrichment of breast cancer stem cells using a keratinocyte serum-free medium.

BACKGROUND: Keratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM. METHODS: A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44(+)/CD24(-) as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin) was analyzed with real-time PCR. RESULTS: Much higher percentage of CSCs was achieved with K-SFM: 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. CONCLUSION: K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

Toxicity of derivatives from semicarbazide-sensitive amine oxidase-mediated deamination of methylamine against Toxoplasma gondii after infection of differentiated 3T3-L1 cells.

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human diseases such as atherosclerosis and diabetes. We report here antimicrobial activities of the metabolites from adipocytes. Specifically, semicarbazide-sensitive amine oxidase of differentiated 3T3-L1 cells was found to utilize methylamine for producing formaldehyde and hydrogen peroxide, accounting for the inhibition of infectivity of Toxoplasma gondii and its replication in these cells. This was demonstrated by the findings that semicarbazide-sensitive amine oxidase was extremely high in differentiated 3T3-L1 cells; and that the infection of these cells by T. gondii and its intracellular replication were decreased to 33% and 37% of the control, respectively, when methylamine was provided in micromolar concentrations as the substrate to the aminoxidase. Only one of the two reaction products expected was found inhibitory against T. gondii when added to the infected pre-adipocytes of 3T3-L1. Intracellular replication of this parasite was inhibited by formaldehyde in the range of 10-100 microM and stimulated by hydrogen peroxide at 1-10 microM. The finding indicates that T. gondii may be useful as a sensitive and convenient sentinel for screening agents toxic to eukaryotic cells.

[Construction of a recombinant lentiviral expression vector carrying carcinoembryonic antigen gene and its expression in dendritic cells].

OBJECTIVE: To construct a lentiviral expression vector of human carcinoembryonic antigen (CEA), and identify its expression in dendritic cells (DCs). METHODS: Human CEA-encoding sequence was amplified, purified, ligated with lentiviral vector plasmid pLentiGFP and verified by sequencing. The verified recombinant vector plasmid (pLentiGFP-CEA), the packaging plasmid p 8.2 and pVSV-G were transfected into 293T cells by Lipofectamine(TM) 2000 reagent. The supernatant of the cultured 293T cells was collected to infect the DCs. The expression of CEA in the transfected DCs was assayed by RT-PCR and Western blotting. RESULTS: CEA lentiviral vector was highly expressed in the transfected DCs as observed using fluorescence microscope 48 h after the the transfection. The human CEA gene was successfully amplified by RT-PCR with a length of about 2100 bp. Western blotting also showed CEA expression in the transfected DCs. CONCLUSION: The human CEA lentiviral expression vector has been successfully constructed and the functional CEA protein can be expression in the transfected DCs. This facilitates further studies of the function of CEA at the molecular level.

[Study on the specific immunity of dendritic cell vaccine loading human umbilical vein endothelial cell antigen against U14 cervical cancer].

AIM: To explore the specific immunity of dendritic cell (DC) vaccine loading human umbilical vein endothelial cell (HUVEC) antigen.against U14 cervical cancer of mice. METHODS: Primary HUVECs were cultured, identified and made into antigen. The BALB/c mice were immunized with DCs loading HUVEC antigen 4 times a week. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, CTL response of spleen lymphocytes in vitro, the percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and Western blot. RESULTS: HUVECs with high purity were successfully cultured and by identified by immunocytochemistry and RT-PCR. After the vaccine was injected into mice, the tumors of mice in PBS group grew faster than the those in other groups. The tumors of mice in HUVEC-DC groups grew slowly and disappeared after 2 weeks and The tumors of mice in DC group disappeared after 3 weeks. The CTL response of spleen lymphocytes in vitro showed that HUVEC-DC-T cells could kill HUVEC cells. The percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes in HUVEC-DC group was higher than that in other groups. The titer of serum antibody was 1:800. immunocytochemistry analysis indicated HUVEC-DC group had specific antigen-antibody reaction to HUVECs through and Western blot analysis revealed there were specific bands at 130 KDa and 220 KDa. CONCLUSION: HUVEC-DC vaccine and DC vaccine can inhibit the tumor growth of U14 cervical cancer of mice. The special cellular and humoral immunity are induced by HUVEC-DC vaccine. Furthermore, some antigens in HUVECs maybe have special immune reaction with integrin alphav and VEGF-R2.