Protein tyrosine kinase 6 is associated with nasopharyngeal carcinoma poor prognosis and metastasis.

BACKGROUND: The aim of this study was to analyze the expression of protein tyrosine kinase 6 (PTK6) in nasopharyngeal carcinoma (NPC) samples, and to identify whether PTK6 can serve as a biomarker for the diagnosis and prognosis of NPC. METHODS: We used quantitative RT-PCR and Western blotting analysis to detect mRNA and protein expression of PTK6 in NPC cell lines and immortalized nasopharyngeal epithelial cell lines. 31 NPC and 16 non-tumorous nasopharyngeal mucosa biopsies were collected to detect the difference in the expression of mRNA level of PTK6 by quantitative RT-PCR. We also collected 178 NPC and 10 normal nasopharyngeal epithelial cases with clinical follow-up data to investigate the expression of PTK6 by immunohistochemistry staining (IHC). PTK6 overexpression on cell growth and colony formation ability were measured by the method of cell proliferation assay and colony formation assay. RESULTS: The expression of PTK6 was higher in most of NPC cell lines at both mRNA and protein levels than in immortalized nasopharyngeal epithelial cell lines (NPECs) induced by Bmi-1 (Bmi-1/NPEC1, and Bmi-1/NPEC2). The mRNA level of PTK6 was high in NPC biopsies compared to non-tumorous nasopharyngeal mucosa biopsies. IHC results showed the expression of PTK6 was significantly correlated to tumor size (P<0.001), clinical stage (P<0.001), and metastasis (P=0.016). The patients with high-expression of PTK6 had a significantly poor prognosis compared to those of low-expression (47.8% versus 80.0%, P<0.001), especially in the patients at the advanced stages (42.2% versus 79.1%, P<0.001). Multivariate analysis indicated that the level of PTK6 expression was an independent prognostic factor for the overall survival of patients with NPC (P <0.001). Overexpression of PTK6 in HNE1 cells enhanced the ability of cell proliferation and colony formation. CONCLUSIONS: Our results suggest that high-expression of PTK6 is an independent factor for NPC patients and it might serve as a potential prognostic biomarker for patients with NPC.

Anti-CDC25B autoantibody predicts poor prognosis in patients with advanced esophageal squamous cell carcinoma.

BACKGROUND: The oncogene CDC25B phosphatase plays an important role in cancer cell growth. We have recently reported that patients with esophageal squamous cell carcinoma (ESCC) have significantly higher serum levels of CDC25B autoantibodies (CDC25B-Abs) than both healthy individuals and patients with other types of cancer; however, the potential diagnostic or prognostic significance of CDC25B-Abs is not clear. The aim of this study is to evaluate the clinical significance of serum CDC25B-Abs in patients with ESCC. METHODS: CDC25B autoantibodies were measured in sera from both 134 patients with primary ESCC and 134 healthy controls using a reverse capture enzyme-linked immunosorbent assay (ELISA) in which anti-CDC25B antibodies bound CDC25B antigen purified from Eca-109 ESCC tumor cells. The clinicopathologic significance of CDC25B serum autoantibodies was compared to that of the tumor markers carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC-Ag) and cytokeratin 19 fragment antigen 21-1(CYFRA21-1). RESULTS: Higher levels of CDC25B autoantibodies were present in sera from patients with ESCC (A450 = 0.917, SD = 0.473) than in sera from healthy control subjects (A450 = 0.378, SD = 0.262, P < 0.001). The area under the receiver operating characteristic (ROC) curve for CDC25B-Abs was 0.870 (95% CI: 0.835-0.920). The sensitivity and specificity of CDC25B-Abs for detection of ESCC were 56.7% and 91.0%, respectively, when CDC25-Abs-positive samples were defined as those with an A450 greater than the cut-off value of 0.725. Relatively few patients tested positive for the tumor markers CEA, SCC-Ag and CYFRA21-1 (13.4%, 17.2%, and 32.1%, respectively). A significantly higher number of patients with ESCC tested positive for a combination of CEA, SCC, CYFRA21-1 and CDC25B-Abs (64.2%) than for a combination of CEA, SCC-Ag and CYFRA21-1 (41.0%, P < 0.001). The concentration of CDC25B autoantibodies in serum was significantly correlated with tumor stage (P < 0.001). Although examination of the total patient pool showed no obvious relationship between CDC25B autoantibodies and overall survival, in the subgroup of patients with stage III-IV tumors, the cumulative five-year survival rate of CDC25B-seropositive patients was 6.7%, while that of CDC25B-seronegative patients was 43.4% (P = 0.001, log-rank). In the N1 subgroup, the cumulative five-year survival rate of CDC25B-seropositive patients was 13.6%, while that of CDC25B-seronegative patients was 54.5% (P = 0.040, log-rank). CONCLUSIONS: Detection of serum CDC25B-Abs is superior to detection of the tumor markers CEA, SCC-Ag and CYFRA21-1 for diagnosis of ESCC, and CDC25B-Abs are a potential prognostic serological marker for advanced ESCC.

[Study on lung carcinogenesis associated genes in human lung squamous cell carcinoma and malignant transformation of human bronchial epithelial cells induced by carcinogen].

OBJECTIVE: To investigate lung carcinogenesis associated genes in human lung squamous cell carcinoma and malignant transformation of human bronchial epithelial cells induced by chemical carcinogens with cDNA microarray. METHODS: The gene expression patterns were detected in all specimens by cDNA microarray which representing 4 096 different human genes. The differences in gene expression among 6 cases of human lung squamous cell carcinoma tissues and 6 normal lung tissues were analyzed. The different gene expression patterns between the normal human bronchial epithelial cell lines (16HBE) and the malignant transformation of human bronchial epithelial cells induced by Benzo(a)pyrene metabolite BPDE (anti-Benzo(a)pyrene diol-epoxide,BPDE) and crystalline nickel sulfide were also studied by that method. The similar changed genes among those gene expression patterns were identified as lung carcinogenesis associated genes. RESULTS: Among the 4096 genes of cDNA microarrays, there were 171 genes expressed differently among lung cancer tissues and normal lungs, 143 genes expressed differently between BPDE transformed cells and normal 16HBE cell lines, 151 genes differed between nickel sulfide transformed cells and normal 16HBE cell lines. By comparing the gene expression profiles, there were 89 similar changed genes which might be associated with human lung carcinogenesis, 39 of which were up regulated: 6 oncogenes, 4 cell cycle control genes, 6 cell proliferation genes, 8 metastasis genes, 3 neuroendocrine genes, 1 drug-resister gene, 1 anti-apoptosis gene, 1 oxidative gene and other 9 genes. 50 genes were down-regulated: 7 tumor suppression genes, 11 DNA repair genes, 1 antioxidant genes, 3 GST family genes, 3 cell framework genes, 2 apoptosis induced genes, 5 signal conduction genes, 5 cytokines and their receptor genes, 7 metabolization genes, 1 cell matrix genes, and other 5 genes. CONCLUSION: cDNA microarray can be applied to study gene expression profiles effectively and to screen human lung carcinogenesis associated genes.

Expression of beclin 1 in bladder cancer and its clinical significance.

BACKGROUND: The aim of this study is to explore the expression of beclin 1, an autophagy gene, in bladder cancer and to evaluate its clinical and prognostic significance in patients with bladder cancer. METHODS: Beclin 1 expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry in bladder cancer tissues and adjacent normal bladder tissues. The relationship between the expression of beclin 1 and clinicopathological characteristics and prognosis was statistically analyzed.
 RESULTS: mRNA level, protein expression and immunoreactivity of beclin 1 were decreased in bladder cancer tissues compared with adjacent normal tissues. Downregulation of beclin 1 was more frequent in tumors with higher histological grades (the expression of beclin 1 was reduced by 49.0% in G1 and G2, and by 71.8% in G3, p=0.010), and was also reduced by 69.5% in the muscle invasive type and by 51.1% in the non-muscle invasive type (p=0.04). Reduced beclin 1 expression was positively associated with higher histological grade and more advanced clinical stage (p<0.05). Kaplan-Meier survival analysis revealed that patients exhibiting lower beclin 1 expression experienced a shorter survival than those with higher expression (p=0.006). Cox proportional hazards regression analysis showed that beclin 1 protein is an independent predictor of survival (p=0.005).
 CONCLUSION: Beclin 1 has an influence on the progression of bladder cancer and might serve as a potential prognostic factor for patients with bladder cancer.

Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

B-lymphoma mouse Moloney leukemia virus insertion region 1 (Bmi1), a member of the polycomb group, has elevated expression and is involved in the pathogenesis of various aggressive cancers, including nasopharyngeal carcinoma (NPC). To date, the mechanisms underlying the high expression of Bmi1 in NPC remain obscure. To gain new insights into the transcriptional regulation of BMI1, we cloned and characterized the promoter region of BMI1. Luciferase reporter assays demonstrated that the region from -783 to +375 showed significant promoter activity. With the use of a series of 5'-deletion and 3'-deletion promoter constructs in luciferase reporter assays, the +167/+232 and -536/-134 regions were found to be sufficient for full promoter activity. Transcriptional activity of the BMI1 promoter was dependent on the Sp1 binding site cluster (+181/+214) as well as the E-box elements (-181), and was abolished after mutation of the two cis-elements. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that Sp1 bound to the region from +181 to +214 within the BMI1 promoter. In addition, gain-of-function and loss-of-function analyses revealed that Sp1 augmented Bmi1 expression. Further investigations using immunohistochemistry and quantitative RT-PCR disclosed a significant positive correlation between the expression of Sp1 and Bmi1 in normal nasopharyngeal epithelial cells, NPC cells, and NPC tissue specimens. In addition, Myc, the known transcription factor for BMI1 in neuroblastomas, also activated the transcription of BMI1 through binding to the E-box element (-181) within its promoter, and showed a positive correlation with the mRNA level of BMI1 in NPC. In conclusion, these findings provide valuable mechanistic insights into the role of Sp1 and c-Myc in BMI1 transcription in NPC, and suggest that targeting of Sp1 or c-Myc may be a potential therapeutic strategy for NPC.

[In vitro induction of mature dendritic cells from malignant pleural effusions].

BACKGROUND & OBJECTIVE: Dendritic cells (DCs) can activate immunologic naive T cells to initiate antigen-specific immune responses. This study was to in vitro induce mature DCs from malignant pleural effusions of patients with lung cancer. METHODS: Malignant pleural effusions (500-1 000 ml) were collected from 16 patients with primary lung cancer. Precursory DCs were obtained through density gradient centrifugation and magnetic cell sorting from malignant pleural effusions, and cultured with interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). Morphology of DCs was observed under invert optical microscope and electronic microscope; phenotype of DCs was analyzed by flow cytometry. Effect of DCs on proliferation of tumor infiltrative lymphocytes (TILs) was observed through mixed lymphocyte reaction (MLR). RESULTS: Mature DCs with typical morphology (elongated dendritic processions observed under invert optical microscope and electronic microscope) were induced from malignant pleural effusions. Expressions of surface phenotypes were higher in DCs induced for 48 h than in DCs induced for 0, 24, 96, and 192 h. When activated with DCs, proliferation of TILs was enhanced. CONCLUSION: Mature DCs could be induced from malignant pleural effusions of patients with lung cancer.

[A study on the role of DNA repair gene O6-methylguanine-DNA methyltransferase in the development of human lung cancer].

OBJECTIVE: To study the role of O(6)-methylguanine-DNA methyltransferase (hMGMT) in the development of human lung cancer. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) method was applied to measure hMGMT mRNA expression in 150 lung cancer specimens, 40 normal lung tissues, and in the peripheral mononuclear blood cells from 50 lung cancer cases and 50 normal controls. The protein expressions of p53, C-MYC and K-RAS were assessed by immuno-histochemistry. The effects of some exposure factors on the expression of hMGMT gene were analyzed. The relationships between hMGMT gene and cancer related genes p53, C-MYC and K-RAS were investigated. RESULTS: The mRNA of hMGMT was low or absent in 49 of 150 (32.7%) lung cancer specimens, whereas 2 of 40 (5%) normal lung tissues had reduced the levels of hMGMT mRNA. The low expression of hMGMT seemed to be a risk factor of lung cancer, with a OR of 9.22 (2.05-57.65). Reduced expression levels of hMGMT mRNA were observed in 10 of 50 (20%) lung cancer patients' peripheral mononuclear blood cells, and 2 of 50 (4%) blood cells among normal controls. When investigating the exposure factors which affecting the expression of hMGMT gene, we noticed that smoking was suppressing the expression of hMGMT gene. Interestingly, over-expression of K-RAS oncogene was significantly correlated with low expression of hMGMT (P < 0.05). However, the expressions of p53 and C-myc were not correlated with the status of hMGMT gene. CONCLUSION: hMGMT might play an important role in the development of human lung cancer. Low expression of hMGMT gene seemed to be a risk factor for lung cancer which could be used as a valuable biomarker on susceptibility of human lung cancers.