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author:("Gong, xiaoke")
1.  TUMOR-SPECIFIC EXPRESSION AND ALTERNATIVE SPLICING OF THE COL6A3 GENE IN PANCREATIC CANCER 
Surgery  2011;150(2):306-315.
Introduction
Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III and V comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked to inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events.
Methods
We analyzed paired PDA-normal (n=18), IPMN (n=5), pancreatic cystadenoma (n=5), and eight PDA cell lines with RT-PCR, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in two animal models of PDA.
Results
COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor-specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in six PDA cell lines, but only two cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells.
Conclusions
We describe, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
doi:10.1016/j.surg.2011.05.011
PMCID: PMC3163121  PMID: 21719059
pancreatic cancer; COL6A3; stroma; microenvironment
2.  Serum Monocyte Chemoattractant Protein-1 in Pancreatic Cancer 
Journal of Oncology  2011;2011:518394.
Background/Aims. Pancreatic ductal adenocarcinoma (PDA) has etiological association with chronic inflammation. Elevated circulating levels of inflammatory mediators, such as monocyte chemoattractant protein-1 (MCP-1), are found in obese individuals. We hypothesized that serum MCP-1 levels are elevated in obese PDA patients. Methods. ELISA was used to analyze MCP-1 serum levels in PDA (n = 62) and intraductal papillary mucinous neoplasms (IPMN) (n = 27). Recursive partitioning statistical analysis investigated the relationship between log MCP-1 and clinicopathological parameters. Results. Log MCP-1 values were significantly (P < 0.05) elevated in patients with BMI ≥ 37.5. In patients with BMI < 37.5, average log MCP-1 values were significantly elevated in PDA patients when compared to IPMN patients. Within the IPMN group, higher log MCP-1 levels correlated with increased age. Recursive partitioning analysis of IPMN versus PDA revealed a strategy of predicting characteristics of patients who are more likely to have cancer. This strategy utilizes log MCP-1 as the primary factor and also utilizes smoking status, gender, and age. Conclusion. MCP-1 is a promising biomarker in pancreatic cancer. The potential of using MCP-1 to distinguish PDA from IPMN patients must be studied in larger populations to validate and demonstrate its eventual clinical utility.
doi:10.1155/2011/518394
PMCID: PMC3184439  PMID: 21977031
3.  INDUCTION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 BY NICOTINE IN PANCREATIC DUCTAL ADENOCARCINOMA CELLS: ROLE OF OSTEOPONTIN 
Surgery  2010;148(2):298-309.
Introduction
Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein- (MCP)-1 and evaluated the role of OPN in mediating these effects.
Methods
MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3–300 nM) or OPN (0.15–15 nM) were analyzed by real time PCR and ELISA. Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-1 transcription. Intracellular and tissue colocalization of OPN and MCP-1 were examined by immunofluorescence and immunohistochemistry.
Results
Nicotine treatment significantly increased MCP-1 expression in PDA cells. Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P<0.05) increased MCP-1 mRNA and protein and induced its promoter activity. MCP-1 was found in 60% of invasive PDA lesions, of which 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA.
Conclusions
Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation through inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects.
doi:10.1016/j.surg.2010.05.002
PMCID: PMC2908036  PMID: 20579680
pancreatic cancer; nicotine; osteopontin; monocyte chemoattractant protein-1
4.  EXPRESSION OF A PRO-METASTATIC SPLICE VARIANT OF OSTEOPONTIN, OPNC, IN HUMAN PANCREATIC DUCTAL ADENOCARCINOMA 
Surgery  2009;146(2):232-240.
Introduction
Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype. We showed recently that nicotine, a major risk factor in pancreatic ductal adenocarcinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supports anchorage independence and maybe the most potent OPN isoform to convey metastatic behavior. In this study, we tested the effect of nicotine on OPNc expression, and analyzed the correlation between total OPN/OPNc levels and patients’ smoking history.
Methods
Real time PCR and UV-light-illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and in PDA cells treated with or without nicotine (3-300 nM). OPN and OPNc were localized by immunohisotchemistry, and ELISA was used to analyze OPN serum levels.
Results
Nicotine treatment of PDA cells selectively induced denovo expression of OPNc. OPNc was found in 87% of invasive PDA lesions, of which 73% were smokers. The levels of OPNc correlated well with higher expression levels of total OPN in the tissue and serum from patients with invasive PDA.
Conclusions
Our data suggest that smoking and nicotine may contribute to PDA metastatic potential through promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined, OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA.
doi:10.1016/j.surg.2009.03.036
PMCID: PMC2777713  PMID: 19628079
pancreatic cancer; nicotine; osteopontin
5.  Hepatitis B Virus-Mediated Changes of Apolipoprotein mRNA Abundance in Cultured Hepatoma Cells 
Journal of Virology  2003;77(9):5503-5506.
An inverse correlation between hepatitis B virus (HBV) and steady-state levels of apolipoprotein AI and CIII mRNAs was observed in two hepatoma cell lines. Analysis of a third line containing an inducible viral genome implicated viral pregenomic RNA in apolipoprotein mRNA reduction. We conclude that HBV alters infected cells despite the absence of overt cytopathogenicity.
doi:10.1128/JVI.77.9.5503-5506.2003
PMCID: PMC153946  PMID: 12692252

Results 1-5 (5)