Significantly upregulated TACSTD2 and Cyclin D1 correlate with poor prognosis of invasive ductal breast cancer.

The tumor-associated calcium signal transducer 2 (TACSTD2) gene has been reported to be highly expressed in many types of human epithelial cancers, and is associated with tumor metastasis and poor prognosis. The aims of the present investigation were to analyze the TACSTD2 and Cyclin D1 expression at the mRNA and protein levels and to assess its prognostic significance in invasive ductal breast cancer (IDC). The expressions of TACSTD2 and Cyclin D1 in IDC tissues were consistently higher than those in the tumor-adjacent non-malignant tissues by a one-step real-time polymerase chain reaction and immunohistochemistry (P<0.001 and P=0.023, respectively). The statistical analysis of clinicopathologic characteristics and immunohistochemistry by the χ(2) test showed that the high expression of TACSTD2 in IDC was correlated to histological grade (P=0.023), P53 status (P=0.042), Cyclin D1 status (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P=0.004) and TNM staging (P<0.001). Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of IDC. These analyses also showed that a high TACSTD2 expression (P=0.003), a high Cyclin D1 expression (P=0.041), and lymph node metastasis (P=0.006) were independent prognosis factors. Collectively, our studies demonstrated that the high expression of TACSTD2 correlates with a poor prognosis in IDC.

A novel LMP1 antibody synergizes with mitomycin C to inhibit nasopharyngeal carcinoma growth in vivo through inducing apoptosis and downregulating vascular endothelial growth factor.

Combined therapy emerges as an attractive strategy for cancer treatment. The aim of this study was to investigate the inhibitory effects of mitomycin C (MMC) combined with a novel antibody fragment (Fab) targeting latent membrane protein 1 (LMP1) on nasopharyngeal carcinoma (NPC) xenograft nude mice. The inhibitory rates of MMC (2 mg/kg), Fab (4 mg/kg), MMC (2 mg/kg) + Fab (4 mg/kg), and MMC (1 mg/kg) + Fab (4 mg/kg) were 20.1%, 7.3%, 42.5% and 40.5%, respectively. Flow cytometry analysis showed that the apoptotic rate of xenograft tumor cells in the MMC and Fab combination group was 28 ± 4.12%, significantly higher than the MMC (2 mg/kg) group (P < 0.01). Immunohistochemical staining showed that VEGF expression in NPC xenografts was significantly inhibited in the combination group compared to the Fab (4 mg/kg) group (P < 0.05). In conclusion, both MMC and Fab could inhibit NPC xenograft tumor growth in vivo and combination therapy showed apparent synergistic anti-tumor effects, which may be due to the induction of tumor cell apoptosis and the downregulation of VEGF expression. These results suggest that the novel combined therapy utilizing traditional chemotherapeutics and antibody-targeted therapy could be a promising strategy for the treatment of NPC.

Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus.

AIM: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity. METHODS: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting, indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo. RESULTS: A recombinant vector was constructed. The Fab was expressed in soluble form in E coli Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL. The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test). CONCLUSION: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

Exosomal miR-222 from adriamycin-resistant MCF-7 breast cancer cells promote macrophages M2 polarization via PTEN/Akt to induce tumor progression.

Exosome-mediated intercellular communication is considered to be an effective mode for malignant cells to transform biological behaviors in stromal cells. However, the mechanisms by which exosomes modulate macrophages within tumor microenvironment remain largely unclear. In this study, we found that both adriamycin-resistant breast cancer (BCa) cells and the corresponding exosomes (A/exo) were capable of inducing macrophages M2 polarization, which promoted the mobility, proliferation, migration and invasion of BCa cells. Since exosomes deliver microRNAs to affect cellular functions in recipient cells, we confirmed that miR-222 was significantly enriched in A/exo and could be successfully transferred to macrophages. Increased miR-222 level was also detected in exosomes derived from plasma and tissues of chemoresistant patients. Moreover, exosomal miR-222 from A/exo polarized M2 macrophages by targeting PTEN and activating Akt signaling pathway, which promoted BCa cells progression in a feed back loop. Co-culture of adriamycin-resistant BCa cells with macrophages in which miR-222 was upregulated or treated with A/exo facilitated tumor growth in vivo. Collectively, our data demonstrated that chemoresistant BCa cells could remodel macrophages within tumor microenvironment by secreting exosomal miR-222, which directly targeted PTEN and caused Akt cascade activation and macrophages M2 polarization. Our findings may provide a foundation for a promising strategy of BCa treatment by targeting exosomes or exosomal miR-222.

[Screening and identification of human anti-c-Met Fab from a phage antibody library].

OBJECTIVE: To screen anti-c-Met Fab from a phage antibody library and identify its binding activity. METHODS: The expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines. RESULTS: c-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence. CONCLUSION: The anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.

Induction of apoptosis by L-NMMA, via FKHRL1/ROCK pathway in human gastric cancer cells.

OBJECTIVE: To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms. METHODS: Apoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting. Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1. Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction. RESULTS: Gastric cancer cells were significantly apoptotic after treatment with N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01). The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253 phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P < 0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P < 0.01) and a significant increase in apoptosis (P < 0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation. CONCLUSIONS: L-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.

Expression of liver insulin-like growth factor 1 gene and its serum level in patients with diabetes.

AIM: To explore the effect of diabetic duration and blood glucose level on insulin like growth factor 1 (IGF-1) gene expression and serum IGF-1 level. METHODS: Diabetes was induced into Sprague Dawley rats by alloxan and then the rats were subdivided into different groups with varying blood glucose level and diabetic duration. The parameters were measured as follows: IGF-1 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), IGF-1 peptide and serum IGF-1 concentration by enzyme-linked immunosorbent assay (ELISA). RESULTS: During early diabetic stage (week 2), in comparison with normal control group (NC), IGF-1 mRNA (1.17 +/- 0.069 vs 0.79 +/- 0.048, P<0.001; 1.17 +/- 0.069 vs 0.53 +/- 0.023, P<0.0005, respectively), IGF-1 peptide contents [(196.66 +/- 14.9) ng/mg(-1) vs (128.2 +/- 11.25) ng/mg(-1), P<0.0005; (196.66 +/- 14.9) ng/mg(-1) vs (74.43 +/- 5.33) ng/mg(-1), P<0.0001, respectively] were reduced in liver tissues of diabetic rats. The IGF-1 gene downregulation varied with glucose control level of the diabetic state, and deteriorated gradually further with duration of diabetes. By month 6, hepatic tissue IGF-1mRNA was 0.71 +/- 0.024 vs 1.12 +/- 0.056, P<0.001; 0.47 +/- 0.021 vs 1.12 +/- 0.056, P<0.0005, respectively. IGF-1 peptide was (114.35 +/- 8.09) ng/mg(-1) vs (202.05 +/- 15.73) ng/mg(-1), P<0.0005; (64.58 +/- 3.89) ng/mg(-1) vs (202.05 +/- 15.73) ng/mg(-1), P<0.0001 respectively. Serum IGF-1 was also lowered in diabetic group with poor control of blood glucose. On week 2, serum IGF-1 concentrations were (371.0 +/- 12.5) ng/mg(-1) vs (511.2 +/- 24.7) ng/mg(-1), P<0.0005, (223.2 +/- 9.39) ng/mg(-1) vs (511.2 +/- 24.7) ng/mg(-1), P<0.0001 respectively. By month 6, (349.6 +/- 18.62) ng/mg(-1) vs (520.7 +/- 26.32) ng/mg(-1), P<0.0005, (188.5 +/- 17.35 vs 520.7 +/- 26.32) ng/mg(-1), P<0.0001, respectively. Serum IGF-1 peptide change was significantly correlated with that in liver tissue (r=0.99, P<0.001). Furthermore, no difference was found in the above parameters between diabetic rats with euglycemia and non-diabetic control group. CONCLUSION: The influence of diabetic status on IGF-1 gene expression in liver tissues is started from early diabetic stage, causing down regulation of IGF-1 expression, and progresses with the severity and duration of diabetic state. Accordingly serum IGF-1 level decreases. This might indicate that liver tissue IGF-1 gene expression is greatly affected in diabetes, thus contributing to reduction of serum IGF-1 level.

Nanoparticles as a vaccine adjuvant of anti-idiotypic antibody against schistosomiasis.

BACKGROUND: The development of new adjuvants for human use has been the focus of attention. This study's aim is to explore the possibility of using nanoparticle Ca nanoparticles (CA) as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis and its protective mechanisms. METHODS: Nanoparticle CA-NP30 conjugate (CA-NP30) was fabricated. BALB/c mice were immunized actively with CA-NP30 to evaluate its effects of protective immunity on mice. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 and the concentrations of IFN-gamma and IL-4 in supernatant of splenocytes were determined via ELISA. RESULTS: Nanoparticle CA could enhance significantly the protective immunity of NP30 against infection of Schistosoma japonicum and the worm reduction rose from 36.0% (NP30 alone) to 52.6%. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 increased remarkably, as compared with those of the group immunized with NP30 alone. The concentration of IFN-gamma in supernatant of splenocyte was drastically elevated [the groups immunized with CA-NP30 and NP30 alone were (493.80 +/- 400.74) pg/ml and (39.03 +/- 39.58) pg/ml, respectively], but the concentration of IL-4 showed no significant difference from that of NP30 alone [(27.94 +/- 9.84) pg/ml vs (27.28 +/- 14.44) pg/ml]. CONCLUSIONS: Nanoparticle CA could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. The mechanism could be that CA-NP30 enhances humoral and cellular immune responses in mice.

[Construction and expression of single chain Fv gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum].

OBJECTIVE: To construct single chain Fv (scFv) gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum. METHODS: The heavy and light chain variable region genes of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum were inserted into two corresponding sites of expression vector pTHA90, and a scFv gene was constructed with a short peptide (Gly4Ser)3 linker gene. The recombinants were determined by digesting with XhoI/SpeI, XbaI/EcoRI and XhoI/EcoRI, and then were introduced into E. coli Top10. The antigen binding activity of expressed product was detected with ELISA. RESULTS: The recombinants were determined by digesting with endonucleases and expected bands were identified. The value of expressed scFv was 3 times higher than negative control by ELISA(OD492 = 1.06). CONCLUSION: The scFv gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was successfully cloned, and the expressed scFv fragment could interact specifically with antigen NP48.

Lentivirus-mediated overexpression of microRNA-199a inhibits cell proliferation of human hepatocellular carcinoma.

microRNA-199a (miR-199a) is a highly conserved miRNA, always deregulated in numerous human tumors. The results of microarray analysis indicated that miR-199a was frequently downregulated in hepatocellular carcinoma (HCC). The expression levels of miR-199a in 11 pairs of matched HCC neoplastic and adjacent non-neoplastic tissues, 5 HCC cell lines and liver cell line L02 were examined by quantitative real-time PCR analysis. We found miR-199a was significantly down-regulated in HCC tissues when compared with pair-matched adjacent non-tumor tissues. We also found the expression level of miR-199a was also substantially decreased in several human HCC cell lines including SMMC-7721, BEL-7402, BEL-7701, MHCC97H, and HepG2. To investigate the role of miR-199a in tumorigenesis, we developed a lentiviral vector for the expression of pre-miR-199a (Lenti-miR-199a). Lenti-miR-199a inhibited HCC cell proliferation in vitro and in vivo. Compared to parental cells or cells transfected with a control vector, the overexpression of microRNA-199a in the HCC cell lines HepG2 stably was showed to reduce cell proliferation in vitro and in vivo. Luciferase reporter assay revealed the regulation of miR-199a on 3'-UTR of HIF-1α. Further investigation confirmed that miR-199a significantly reduced the endogenous protein level of HIF-1α in hypoxia. MiR-199a inhibits cell proliferation in vitro and in vivo partly through down-regulation of HIF-1α in human HCC. Thus, these studies provide an important new insight into the pathogenesis of human HCC and it may open a new perspective for the development of effective gene therapy for human HCC.

Alpha B-crystallin is a new prognostic marker for laryngeal squamous cell carcinoma.

BACKGROUND: Alpha B-crystallin (αB-crystallin) has been suggested to play an important role in the development of solid tumors. However, the association between αB-crystallin expression and clinicopathological characteristics of human laryngeal carcinoma is not well defined. This study aimed to examine the expression of αB-crystallin in human laryngeal squamous cell carcinoma (LSCC) and investigate the relationship between its expression and the prognosis of LSCC. METHODS: Real-time polymerase chain reaction (six LSCC samples, six tumor-adjacent normal samples) and immunohistochemistry by tissue microarrays (109 LSCC samples and 28 tumor-adjacent normal samples) were performed to characterize expression of the αB-crystallin gene in LSCC. Kaplan-Meier survival and Cox regression analyses were carried out to evaluate the prognosis of LSCC. RESULTS: Real-time polymerase chain reaction and immunohistochemistry analysis showed that the expression of αB-crystallin in LSCC was significantly higher than that in tumor-adjacent normal tissues. Moreover, the expression level of αB-crystallin protein in LSCC was significantly related to alcohol consumption (P = 0.022), tumor differentiation (P = 0.007), pTNM stage (P = 0.041) and 5 years' survival (P =0.030). COX multi-factor analysis showed that αB-crystallin (P = .013), as well as pTNM stage (P =0.027) and lymphatic metastasis (P = 0.015) were independent prognosis factors for LSCC. CONCLUSIONS: The data suggest that αB-crystallin expression is correlated with malignant phenotypes of LSCC and it may serve as a novel prognostic factor for LSCC.

Expression, characterization and therapeutic efficacy of chimeric Fab of anti-idiotypic antibody NP30 against Schistosoma japonicum.

The murine monoclonal anti-idiotypic antibody NP30 is a promising therapeutic antibody against Schistosoma japonicum. However, the immunogenicity of murine NP30 limits its further study and application in humans. Here the chimeric Fab of NP30 (chFab-NP30) comprising the variable regions of murine NP30 and constant regions of human antibody was assembled. chFab-NP30 was expressed and purified as a soluble and functional protein. Administration of chFab-NP30 in vivo increased the survival rate, reduced egg burdens and ameliorated organ pathology of mice with acute schistosomiasis. Our study indicated that chFab-NP30 is a promising candidate to be used as a specific and efficient recombinant antibody against acute schistosomiasis japonica. Further studies on function mechanism of chFab-NP30 needs to be carried out in the future.

Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND: We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma (HCC) cells and its effect on cell growth. METHODS: Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGF gene was designed, synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance (ANOVA) for multiple groups. RESULTS: Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%). CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5 - 8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P < 0.05). Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P < 0.05). The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P < 0.05). CONCLUSIONS: CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.

Evaluation of an IgY-based immunomagnetic enzyme-linked immunosorbent assay system for detection of circulating Schistosoma japonicum antigen in serum samples from patients in China.

We have developed a novel egg yolk antibody (IgY)-coated magnetic beads antigen-capture immunoassay for detection of a circulating antigen of Schistosoma japonicum in serum samples of patients in schistosomiasis-endemic areas of China. This IgY-based immunomagnetic bead enzyme-linked immunosorbent assay (IgY-IMB-ELISA) uses polyclonal IgY-coated magnetic beads as a capture antibody, and a monoclonal IgG as a detection antibody. The sensitivity of the magnetic immunoassay was 100% (40 of 40) in cases of acute infection and 91.5% (107 of 117) in chronic cases of schistosomiasis, and no positive reaction was found in 0 of 49 healthy persons. Cross-reactivity was 3.3% (1 of 33) with clonorchiasis and 0% (0 of 20) with paragonimiasis. There was a significant correlation between ELISA absorbance value and egg count (eggs per gram feces) and a correlation coefficient of 0.88 in a small sample of 14 patients. The results demonstrated that the IgY-IMB-ELISA is a sensitive and specific assay for detection of human schistosomiasis japonica.

Expression and prognostic significance of the alpha B-crystallin gene in human hepatocellular carcinoma.

The aim of this study was to characterize expression of the alpha B-crystallin gene in human hepatocellular carcinomas, to investigate the relationship between expression of this gene and the prognosis of human hepatocellular carcinoma. Real-time polymerase chain reaction, reverse transcriptase-polymerase chain reaction and immunohistochemistry were used to characterize expression of the alpha B-crystallin gene in human hepatocellular carcinoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of human hepatocellular carcinoma. We characterized alpha B-crystallin gene expression in human hepatocellular carcinoma. Statistical analysis of hepatocellular carcinoma patients showed that patients expressing alpha B-crystallin have different survival rates relative to those not expressing this gene (P = .041). After 18 months, the survival rate of patients expressing alpha B-crystallin declined, but survival in the alpha B-crystallin-negative group remained stable. COX multi-factor analysis showed that alpha B-crystallin (P = .007) and venous invasion (P = .037) were independent prognosis factors for hepatocellular carcinoma. Expression of the alpha B-crystallin gene, which is related with the transferability and invasive capacity of hepatocellular carcinoma cells, can be used as a prognostic indicator in human hepatocellular carcinomas. It may also be involved in the malignant transformation of hepatocytes.

Characteristic gene expression profiles in the progression from liver cirrhosis to carcinoma induced by diethylnitrosamine in a rat model.

BACKGROUND: Liver cancer is a heterogeneous disease in terms of etiology, biologic and clinical behavior. Very little is known about how many genes concur at the molecular level of tumor development, progression and aggressiveness. To explore the key genes involved in the development of liver cancer, we established a rat model induced by diethylnitrosamine to investigate the gene expression profiles of liver tissues during the transition to cirrhosis and carcinoma. METHODS: A rat model of liver cancer induced by diethylnitrosamine was established. The cirrhotic tissue, the dysplasia nodules, the early cancerous nodules and the cancerous nodules from the rats with lung metastasis were chosen to compare with liver tissue of normal rats to investigate the differential expression genes between them. Affymetrix GeneChip Rat 230 2.0 arrays were used throughout. The real-time quantity PCR was used to verify the expression of some differential expression genes in tissues. RESULTS: The pathological changes that occurred in the livers of diethylnitrosamine-treated rats included non-specific injury, fibrosis and cirrhosis, dysplastic nodules, early cancerous nodules and metastasis. There are 349 upregulated and 345 downregulated genes sharing among the above chosen tissues when compared with liver tissue of normal rats. The deregulated genes play various roles in diverse processes such as metabolism, transport, cell proliferation, apoptosis, cell adhesion, angiogenesis and so on. Among which, 41 upregulated and 27 downregulated genes are associated with inflammatory response, immune response and oxidative stress. Twenty-four genes associated with glutathione metabolism majorly participating oxidative stress were deregulated in the development of liver cancer. There were 19 members belong to CYP450 family downregulated, except CYP2C40 upregulated. CONCLUSION: In this study, we provide the global gene expression profiles during the development and progression of liver cancer in rats. The data obtained from the gene expression profiles will allow us to acquire insights into the molecular mechanisms of hepatocarcinogenesis and identify specific genes (or gene products) that can be used for early molecular diagnosis, risk analysis, prognosis prediction, and development of new therapies.

[Construction of pMSCV recombinant retroviral vector containing polo-like kinase 3(plk3) cDNA and its effects on cell proliferation].

AIM: To construct pMSCV retroviral vector (RV) containing Polo-like kinase 3(plk3) cDNA, a new member of Ser/Thr protein kinase family, and to study the function of plk3 gene by introducing it into human K562 and HL60 cells. METHODS: plk3 cDNA was sub-cloned into retroviral vector pMSCV-puroR to generate pMSCV-plk3-puroR, pMSCV-puroR being used as the control. RVs were generated by introducing RV vectors into 293T-Amphotropic packaging cells. Titers of RV were measured by NIH3T3 cells with Large-scale-real-time titration method (LaSRT). K562 and HL60 cells were infected with pMSCV-plk3-puroR or pMSCV-puroR viral supernatant by flow-through transduction method, respectively. Following 3-day selection with 3 mg/L puromycin, K562-plk3-puroR, HL60-plk3-puroR, K562-puroR and HL60-puroR cells were obtained. PCR was used to identify the integration of plk3 gene in K562-plk3-puroR and HL60-plk3-puroR cells. Transduction rates were determined by clone-forming ability against puromycin in semi-solid culture medium. Cell growth curve was measured by MTT colorimetry, the cell cycle and apoptotic analysis were detected by flow cytometry, etc. RESULTS: Virus titers generated was (1.31+/-0.65)x10(9)TU/L. The K562 and HL60 transfection efficiency reached 82.3%+/-3.70% and 62.9%+/-4.94%, respectively (n=3). After 3 d puromycin selection, purified K562-plk3-puroR and HL60-plk3-puroR cell were obtained. The proliferation of K562 and HL60 cells was slowed down by plk3 gene transduction. Compared with wild type K562 cells: (1) when cultured in complete DMEM medium, more K562-plk3-puroR cells were observed in G(0)-G(1), 49.7%+/-3.38% vs 43.9%+/-2.34% (P=0.03); (2) when cultured in serum-free DMEM medium for 10 h, fewer K562-plk3-puroR cells were observed in S phase, 43.6%+/-3.74% vs 54.5%+/-1.52% (P=0.02) with lower apoptosis rate, 3.4%+/-0.37% vs 1.3%+/-0.31% (P=0.01). CONCLUSION: Introduction of plk3 gene into tumor cells with RV vectors can slow down cell proliferation, prevent cells into cell cycle and protect cells from apoptosis in serum-free culture.

[Correlation of cyclin D1 overexpression to mutations of von hippel-lindau gene in renal clear cell carcinoma].

BACKGROUND & OBJECTIVE: The mutation of von Hippel-Lindau (VHL) tumor suppressor gene is closely related with tumorigenesis of renal clear cell carcinoma (RCCC). Cyclin D1 gene plays an important role in progression of RCCC by stimulating cell proliferation. This study was to determine the mutation of VHL gene in RCCC, and explore its correlation to overexpression of Cyclin D1. METHODS: The specimens of RCCC and adjacent normal renal tissue from 50 patients were collected after surgery. Total RNA and genomic DNA were extracted from each sample. Variant exons in VHL gene were amplified by polymerase chain reaction (PCR) and sequenced, and DNA hypermethylation was detected by restriction analysis. Reverse transcription-PCR (RT-PCR) and Western blot were used to detect the expression of Cyclin D1. RESULTS: Of the 50 specimens, 42 (84.0%) had various VHL gene mutations, 12 (24.0%) had more than 1 kind of gene mutation. Of the 57 cases of exon mutation of VHL gene, 17 (29.8%) were located in exon 1, 26 (45.6%) in exon 2, and 14 (24.6%) in exon 3. The expression of Cyclin D1 in the 42 cases with VHL gene mutation was increased to 2-10 (3.91+/-1.54) times that of normal controls (P<0.01). Cyclin D1 expression in the other 8 cases was normal. CONCLUSION: There are variant mutations of VHL gene in RCCC, which may lead to overexpression of Cyclin D1.

[The expression of hTERT and p53 protein in pre-malignant and malignant lesions of human oral mucosa].

PURPOSE: To investigate the roles of hTERT and p53 protein in malignant changes of oral mucosal precancerous lesions and the relations between hTERT and p53. METHODS: The expression of hTERTmRNA and p53 were measured using in situ hybridization and immunohistochemical assay in 9 cases of oral mucosal hyperplasia, 11 cases of light dysplasia, 10 cases of medium dysplasia, 9 cases of carcinomas in situ and 11 cases of squamous cell carcinomas. The results were processed by medical photographic system, and analyzed statistically by one-way ANOVA. The relation to hTERT and p53 was analyzed statistically by Pearson correlations. RESULTS: It was found that from hyperplasia to light dysplasia, medium dysplasia, carcinomas in situ and squamous cell carcinomas, the indexes of positive cell vessels and the value of OD increased, arriving at the apex in squamous cell carcinoma. With phenotypic progression and the degree of dysplasia, hTERT and p53 expression cells extend from basal layer to the keratinous layer. In highly-differentiated squamous cell carcinomas, expression cells were mainly located around the nests. Stained cells dispersed throughout the tumor tissue in low-differentiated squamous cell carcinomas. p53 was undetectable in hyperplasia and light dysplasia, but p53 was observed in 66.67% cases of high dysplasia and 72.72% cases of squamous cell carcinomas. There was not a significant linear correlation between hTERT and p53. CONCLUSIONS: The results suggest that activation of telomerase and p53 play a role in the process of malignant changes of oral mucosal precancerous lesions. Oral squamous cell carcinoma is a disease that has been found in association with many factors.

[The expression of apoptosis-associated proteins Bcl-2, Bax in oral leukoplakia and lichen planus].

PURPOSE: The purpose of this study was to explore the pathogenesis and carcinogenesis of oral leukoplakia (LK) and oral lichen planus (OLP) by examining the expression of apoptosis-associated proteins Bcl-2 Bax in LK and OLP. METHODS: The expression of Bcl-2 and Bax were measured in 10 cases of normal oral mucosa,18 cases of OLP, 23 cases of LK and 22 cases of oral squamous cell carcinoma (SCC) by immunohistochemical assays. RESULTS: In the epithelial cell layer of LK and OLP, the positive Bcl-2 expression was similar to oral normal mucosa,but in the part of the lymphocytic infiltration of OLP, overexpression of Bcl-2 was observed. In the SCC, the Bcl-2 expression was significantly higher than that in normal oral mucosa(P<0.05). In the tissue of simple hyperplasia, mild dysplasia, moderate dysplasia, poorly differentiated SCC and erosive OLP, the expression of Bax was significantly higher than that in normal oral mucosa (P<0.05). CONCLUSIONS: Bax was closely related to the early event of LK carcinogenesis. Bcl-2 may not play a role in LK carcinogenesis. Bcl-2 and Bax play an important role in pathogenesis of OLP. We postulate that the Bcl-2 inhibits the apoptosis of lymphocytes that strengthen the cell-mediated immune process and the overexpression of Bax was related to the apoptosis of epithelial cells in OLP.