T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN- production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN- T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.     

Wnt control of stem cells and differentiation in the intestinal epithelium

The intestinal epithelium represents a very attractive experimental model for the study of integrated key cellular processes such as proliferation and differentiation. The tissue is subjected to a rapid and perpetual self-renewal along the crypt-villus axis. Renewal requires division of multipotent stem cells, still to be morphologically identified and isolated, followed by transit amplification, and differentiation of daughter cells into specialized absorptive and secretory cells. Our understanding of the crucial role played by the Wnt/beta-catenin signaling pathway in controlling the fine balance between cell proliferation and differentiation in the gut has been significantly enhanced in recent years. Mutations in some of its components irreversibly lead to carcinogenesis in humans and in mice. Here, we discuss recent advances related to the Wnt/beta-catenin signaling pathway in regulating intestinal stem cells, homeostasis, and cancer. We emphasize how Wnt signaling is able to maintain a stem cell/progenitor phenotype in normal intestinal crypts, and to impose a very similar phenotype onto colorectal adenomas.     

Mechanisms of mitochondrial apoptosis induced by peripheral benzodiazepine receptor ligands in human colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce apoptosis in gastrointestinal cancers. The aim of this study was to characterize the signaling pathways of PBR ligand-induced apoptosis. FGIN-1-27 but not PK 11195-induced apoptosis was associated with a decrease of mitochondrial membrane potential and an increase of mitochondrial volume in HT29 colorectal cancer cells. However, PK 11195-elicited apoptosis was associated with a downregulation of Bcl-2, translocation of Bax to the mitochondria including subsequent oligomerization, and activation of caspase-9, indicating the involvement of mitochondria in PK 11195-induced apoptosis. Moreover, PK 11195-induced apoptosis was associated with the generation of reactive oxygen species. This study demonstrates a novel mechanism of PK 11195-induced mitochondrial apoptosis without alteration of the mitochondrial membrane potential. The characterization of signaling pathways associated with PBR ligand-induced apoptosis will build the base for a future use of these ligands in anti-neoplastic therapeutic approaches.     

A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C-->T transition at -1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 -1306 C-->T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055-3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT+TT genotypes. Our data suggest that MMP-2 -1306 C-->T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.     

甘油二酯激酶α在结直肠癌中的表达及其与PKC、TNFα的相关性

目的探讨甘油二酯激酶α(DGKα)在结直肠癌中的表达及其与蛋白激酶C(PKC)、肿瘤坏死因子α(TNFα)表达的相关性。方法应用免疫组化方法检测DGKα、PKC和TNFα在48例结直肠癌、癌旁正常组织和9例腺瘤性息肉中的表达。结果 DGKα在结直肠癌组织和癌旁正常组织有表达,结直肠癌组织中DGKα阳性表达率(79.2%)显著高于腺瘤性息肉(阴性)和癌旁正常组织(33.3%,P0.05);PKC主要分布在结直肠癌和癌旁正常组织中,结直肠癌组织中PKC阳性表达率(35.4%)显著高于腺瘤性息肉(阴性),与癌旁正常组织(20.8%)相比无显著性差异(P0.05);TNFα在三种组织中均表达阳性,结直肠癌组织中TNFα阳性表达率(95.8%)显著高于腺瘤性息肉(55.6%,P0.05),与癌旁正常组织(87.5%)相比无显著性差异(P0.05);结直肠癌组织中,DGKα与PKC的表达呈负相关(r=-0.437,P0.05),与TNFα的表达没有相关性(r=0.185,P0.05)。结论DGKα在结直肠癌组织中的表达高于腺瘤性息肉,DGKα可能抑制了PKC的活性,但对TNFα没有明显的抑制作用,在临床病理鉴别诊断中有辅助价值。     

Monoclonal antibody A7 coupled to magnetic particles as a contrast enhancing agent for magnetic resonance imaging of human colorectal carcinoma

Background: Local recurrence, the most frequent pattern of recurrence of rectal carcinoma, is almost always fatal. The difficulty of diagnosing local recurrence contributes importantly to the poor prognosis. Methods: We coupled monoclonal antibody (Mab) A7, which reacts specifically with human colorectal carcinoma, to ferromagnetic lignosite (FML) particles to distinguish rectal carcinoma from other tissues by magnetic resonance (MR) imaging. We examined retention of immunoreactivity by the A7-FML complexes in vitro, and also their distribution in vivo according to radiolabeling and MR imaging when injected into nude mice bearing human colorectal carcinoma xenografts. Results: A7-FML retained binding activity nearly identical to that of Mab A7. Significantly more ¹²⁵I-labeled A7-FML accumulated in engrafted tumors than did ¹²⁵I-labeled normal mouse IgG-FML complexes (P<0.05). A7-FML disappeared rapidly from the blood. Normal tissues accumulated less ¹²⁵I-labeled A7-FML than tumors; this accumulation decreased linearly with time. In MR imaging, signal intensity was reduced in the tumor by the injection of A7-FML. Conclusions: A7-FML is potentially useful as a MR contrast enhancing agent for human colorectal carcinoma xenografts implanted subcutaneously.     

The CpG island methylator phenotype in colorectal cancer: Progress and problems

In recent years, attention has focused on the biology and potential clinical importance of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). While it is generally well accepted that etiologically and clinically distinct subgroups exist in this disease, a precise definition of CIMP remains to be established. Here, we summarize existing literature that documents the prevalence of CIMP in CRC, with particular attention to the various methods and definitions used to classify a tumor as CIMP positive. Through a systematic review on both case-series and population based studies, we examined only original research articles reporting on sporadic CRC and/or adenomas in unselected cases. Forty-eight papers published between January 1999 and August 2011 met the inclusion criteria. We describe the use of multiple gene panels, marker threshold values, and laboratory techniques which results in a wide range in the prevalence of CIMP. Because there is no universal standard or consensus on quantifying the phenotype, establishing its true prevalence is a challenge. This bottleneck is becoming increasingly evident as molecular pathological epidemiology continues to offer possibilities for clear answers regarding environmental risk factors and disease trends. For the first time, large, unselected series of cases are available for analysis, but comparing populations and pooling data will remain a challenge unless a universal definition of CIMP and a consensus on analysis can be reached, and the primary cause of CIMP identified.     

Analysis of O6-methylguanine-DNA methyltransferase methylation status in sporadic colon polyps

Background/aimThe aim of our study was to check how MGMT methylation status together with known factors influenced the risk of colon cancer development.Materials and methodsWe examined patients with colon polyps. Information concerning gender, age, lifestyle, diet, anthropometry and medical information, including cancer and family history of cancer, was analyzed. Polymorphism variety of MGMT gene was investigated in another study. Genetic analysis for MGMT methylation assessment was performed for polyp tissue samples from 143 patients.ResultsPositive methylation MGMT status was found in 55 patients. There was no correlation between gender and MGMT methylation status (p = 0.43). We did not find correlation between patients younger and older than 60 (p = 0.87). There was no correlation between smoking and MGMT methylation status (p = 0.36). We did not find correlation between BMI and MGMT methylation status (p = 0.86). We did not find correlation between MGMT methylation status and colon cancer in familial history (p = 0.45).ConclusionOur study showed no correlations between methylation status of MGMT polymorphisms and clinical features like age, gender, polyp localization, smoking status, or obesity. It has been shown previously that MGMT methylation status may show nonspecific methylation in colon polyps. Gene methylation status in adenoma tissues has also been associated by other authors with the adenoma's size, histology, and degree of atypia. In our study, we evaluated the gene methylation status in colon polyps and found no association with adenoma characteristics. The present study showed no correlation for MGMT methylation in polyps in different regions of colon.     

Effects of siRNA Targeting c‐Myc and VEGF on Human Colorectal Cancer Volo Cells

c‐Myc and vascular endothelial growth factor (VEGF) genes are frequently deregulated and overexpressed in this malignancy, and strategies designed to inhibit c‐Myc and VEGF expression in cancer cells may have considerable therapeutic value. In the present study, we design and use short interfering RNA (siRNA) to inhibit c‐Myc and VEGF expression in colorectal cancer Volo cells and validate their effects on cell proliferation, cell cycle, apoptosis, and cell metastasis. Upon transient transfection with plasmid‐encoding siRNA, it was found that expression of c‐Myc and VEGF was significantly downregulated in siRNA‐transfected cells and the downregulation of c‐Myc and VEGF inhibited cell growth and induced apoptosis and metastasis of Volo cells. c‐Myc and VEGF downregulation also increased cell population in the G0–G1 phase. In conclusion, the specific siRNA efficiently silenced the expression of c‐Myc and VEGF, further suppressed the cell proliferation, triggered cell apoptosis, and inhibited cell invasiveness of colorectal cancer Volo cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:499‐505, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21455