Colon Adenocarcinoma Tissues Research Articles

Decreased expression of 17β-hydroxysteroid dehydrogenase type 1 is associated with DNA hypermethylation in colorectal cancer located in the proximal colon

BackgroundThe importance of 17β-estradiol (E2) in the prevention of large bowel tumorigenesis has been shown in many epidemiological studies. Extragonadal E2 may form by the aromatase pathway from androstenedione or the sulfatase pathway from estrone (E1) sulfate followed by E1 reduction to E2 by 17-β-hydroxysteroid dehydrogenase (HSD17B1), so HSD17B1 gene expression may play an important role in the production of E2 in peripheral tissue, including the colon.MethodsHSD17B1 expression was analyzed in colorectal cancer cell lines (HT29, SW707) and primary colonic adenocarcinoma tissues collected from fifty two patients who underwent radical colon surgical resection. Histopathologically unchanged colonic mucosa located at least 10-20 cm away from the cancerous lesions was obtained from the same patients. Expression level of HSD17B1 using quantitative PCR and western blot were evaluated. DNA methylation level in the 5' flanking region of HSD17B1 CpG rich region was assessed using bisulfite DNA sequencing and HRM analysis. The influence of DNA methylation on HSD17B1 expression was further evaluated by ChIP analysis in HT29 and SW707 cell lines. The conversion of estrone (E1) in to E2 was determined by electrochemiluminescence method.ResultsWe found a significant decrease in HSD17B1 transcript (p = 0.0016) and protein (p = 0.0028) levels in colorectal cancer (CRC) from the proximal but not distal colon and rectum. This reduced HSD17B1 expression was associated with significantly increased DNA methylation (p = 0.003) in the CpG rich region located in the 5' flanking sequence of the HSD17B1 gene in CRC in the proximal but not distal colon and rectum. We also showed that 5-dAzaC induced demethylation of the 5' flanking region of HSD17B1, leading to increased occupation of the promoter by Polymerase II, and increased transcript and protein levels in HT29 and SW707 CRC cells, which contributed to the increase in E2 formation.ConclusionsOur results showed that reduced HSD17B1 expression can be associated with DNA methylation in the 5' flanking region of HSD17B1 in CRC from the proximal colon.

An evaluation of CD133 as a biomarker for malignant transformation of colon polyps.

e14045 Background: CD133 is a pentaspan membrane glycoprotein expressed by blood and neuronal stem cells but its biological role in colon cancer remains controversial. The expression of CD133 in normal colonic tissue and benign and malignant polyps has not been studied in a progressive manner. We hypothesize that expression of CD133 increases as the polyp progresses from non-cancerous to adenocarcinoma of the colon. Methods: Archived normal colonic epithelium and non-cancerous polyps as well as adenocarcinomas were evaluated immunohistochemically using CD133 antibody. A grading scale was used to classify staining patterns as negative (no staining) or positive (any staining). Positive staining was further classified as low (<50%) and high (>50%) glandular staining as described previously. Results: A gradation of CD133 expression was noted with almost no expression in normal colon (100% negative, n = 5) to higher expression, gradually increasing from hyperplastic polyps to adenocarcinoma. In the hyperplastic polyps (n=12), 9 were classified as negative and 3 classified as positive (25%) with 1 having high and 2 having low expression. In the tubular polyps (n=18), all 18 samples were positive (100%), with 10 high expression and 8 low. In the villous polyps (n=7), all 7 samples were positive (100%) with high expression. Finally, with colonic adenocarcinoma tissue (n=12), all 12 samples had positive expression (100%) for CD133 with 8 having high expression and 4 low expression. Most interestingly the expression of CD133 in the samples examined increased from 25% positive in benign hyperplastic polyps to 100% positive in tubular polyps indicating the potential importance of CD133 as an early biomarker for malignant transformation. Conclusions: Our results indicate a gradual increase in CD133 expression in the progression from normal colonic epithelium, to hyperplastic, to tubular/villous polyps and adenocarcinoma, which displayed the highest expression (Cochran-Armitage test for trend, p <.0001). Taken together, our studies indicate that expression of CD133 may serve as an early bio-marker for malignant transformation and provide a platform to examine the potential benefit of antibody based therapy in patients with colon cancer.

Abstract 3920: Oxidatively-induced complex DNA damage and repair in human colon adenocarcinoma

Abstract Colorectal cancer is the third leading cause of cancer mortality in the United States. Oxidative stress and DNA damage have been associated with a variety of human pathophysiological conditions, including colon cancer. Complex DNA damage may manifest in double strand breaks (DSBs) and non-DSB, bi-stranded, oxidatively-induced clustered DNA lesions (OCDLs). To date, there is very limited knowledge regarding the possible accrual of OCDLs in human colon tumors or the OCDL repair efficiency of primary colonic malignant cells. Therefore, the impact of oxidatively-induced clustered DNA damage in the predisposition to or progression of colon cancer remains undefined. We hypothesized that colon adenocarcinoma tissue and cells would have increased levels of DNA damage compared with normal counterparts. Furthermore, we expected that malignant colon cells would exhibit a defective Base Excision Repair (BER) pathway. To test this hypothesis, normal and malignant colonic tissue from 10 consented female patients was collected and number of DNA lesions measured using neutral constant field gel electrophoresis. Quantitative measurement of OCDL difference in each group was achieved using QuantiScan analysis. DNA repair efficiency of colon tissue-derived primary normal and malignant cells was assessed by single-cell gel electrophoresis. Expression and localization of BER-associated proteins was determined through Western blot analysis and immunohistochemistry. We found that degree of complex DNA damage varied among individual subjects. Forty percent of patients had tumors with increased levels of APE1-induced OCDLs compared to normal counterparts. The vast majority (80%) of malignant tissue expressed higher levels of APE1, XRCC1 and PARP1, ranging from 3- to &amp;gt;10-fold, compared to normal peri-tumoral tissue. However, 20% of malignant tissue had reduced levels of the aforementioned proteins as compared to peri-tumoral matched normal tissue. Oxidatively-induced DNA damage was more efficiently repaired in primary cells derived from normal tissue compared to cells from paired malignant tissue (1 hour versus &amp;gt;6 hours) following treatment with 6 mM H2O2. Since the expression of most BER-associated proteins (e.g., XRCC1, OGG1, etc.) was similar in both malignant and normal cells, the DNA repair defect may be the result of a 2.5-fold lower expression of APE1 protein measured in these cancerous cells compared to normal primary cells. Furthermore, a differential cytoplasmic to nuclear translocation of APE1 was also observed in cancerous cells compared to normal cells during the DNA damage and repair process. In conclusion, these findings strongly indicate that there is variation in oxidatively-induced complex DNA damage – perhaps mediated through a defective BER pathway – among patients with colon cancer, thus underscoring the need for patient-specific approaches to diagnosis and treatment of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3920. doi:10.1158/1538-7445.AM2011-3920

VEGF-A not Ang2 mediates endothelial-like differentiation of immature DCs by ERK1/2 signaling in the microenvironment of human colon adenocarcinoma

Endothelial-like differentiation of dendritic cells (DCs) is an interesting and significant phenomenon, which is worth further investigation. Here, we show that the tumor microenvironment derived from the supernatant of the SW620 human colon adenocarcinoma cell line and colon adenocarcinoma tissue homogenate can promote immature DCs (iDCs) to differentiate from the DC pathway toward endothelial cells, while the peri-carcinoma homogenate supernatant does not have this role. Inhibition of angiopoietin-2 (Ang2) in the supernatant by its antibody has no obvious influence on the endothelial-like differentiation. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) blocked the differentiation. During the course of differentiation, a sustained activation of ERK1/2 was detected. PD98059 blocked the phosphorylation of ERK1/2 as well as the endothelial-like differentiation of iDCs. Inhibition of VEGF-A also blocked the phosphorylation of ERK1/2. These data suggest that VEGF-A not Ang2 mediates endothelial-like differentiation of iDCs by ERK1/2 signaling in the microenvironment of human colon adenocarcinoma.

Up-regulated expression of Notch1 and Jagged1 in human colon adenocarcinoma

Deregulated expression of Notch molecules is observed in many malignant tumors, however, the expression of Notch1 and Jagged1 in colon adenocarcinoma is still unknown. This study is to investigate the expression of Notch1 and Jagged1 in human colon adenocarcinoma. Sixty-five human colon adenocarcinoma and 60 adjacent nontumor colon tissue sections were detected by immunohistochemistry. Ten paired fresh surgical human colon adenocarcinoma and adjacent nontumor colon samples were analyzed by Western blot and RT-PCR. Both Notch1 and Jagged1 were expressed in the cytoplasm of neoplastic cells of colon adenocarcinoma tissue. The protein and mRNA levels of both molecules were higher in colon adenocarcinoma than in adjacent nontumor tissue. Moreover, Notch1 was positively correlated with tumor stage. This investigation demonstrates that Notch1 and Jagged1 are up-regulated in human colon adenocarcinoma and suggests that Notch1/Jagged1 signaling might play a role in the development of colon adenocarcinoma.

Relationship and prognostic significance of SPARC and VEGF protein expression in colon cancer

BackgroundSPARC (secreted protein, acidic and rich in cysteine) is closely related with the progress, invasion and metastasis of malignant tumor and angiogenesis.MethodsUsing human colon adenocarcinoma tissues (hereinafter referred to as colon cancer) and their corresponding non-diseased colon from 114 patients' biopsies, the expression of SPARC and vascular endothelial growth factor (VEGF) were investigated by immunohistochemistry staining to assessment the relationship between SPARC and VEGF, as well as their prognostic significance in patients. Evaluation of VEGF expression level with the same tissues was used to establish the antigenic profiles, and the marker of CD34 staining was used as an indicator of microvessel density (MVD).ResultsSPARC expression was mainly in the stromal cells surrounding the colon cancer, and was significant difference in those tissues with the lymph node metastasis and differentiation degree of tumor. Expression of SPARC was significantly correlated with the expression of VEGF and MVD in colon cancer tissues. Patients with low or absence expressing SPARC had significantly worse overall survival and disease-free survival in a Single Factor Analysis; Cox Regression Analysis, SPARC emerged as an overall survival and disease-free survival independent prognostic factor for colon cancer.ConclusionThe low expression or absence of stromal SPARC was an independent prognostic factor for poor prognosis of colon cancer. SPARC maybe involved in the regulation of anti-angiogenesis by which it may serve as a novel target for colon cancer treatment as well as a novel distinctive marker.

Syndecan-2 Functions as a Docking Receptor for Pro-matrix Metalloproteinase-7 in Human Colon Cancer Cells

Although elevated syndecan-2 expression is known to be crucial for the tumorigenic activity in colon carcinoma cells, how syndecan-2 regulates colon cancer is unclear. In human colon adenocarcinoma tissue samples, we found that both mRNA and protein expression of syndecan-2 were increased, compared with the neighboring normal epithelium, suggesting that syndecan-2 plays functional roles in human colon cancer cells. Consistent with this notion, syndecan-2-overexpressing HT-29 colon adenocarcinoma cells showed enhanced migration/invasion, anchorage-independent growth, and primary tumor formation in nude mice, paralleling their morphological changes into highly tumorigenic cells. In addition, our experiments revealed that syndecan-2 enhanced both expression and secretion of matrix metalloproteinase-7 (MMP-7), directly interacted with pro-MMP-7, and potentiated the enzymatic activity of pro-MMP-7 by activating its processing into the active MMP-7. Collectively, these data strongly suggest that syndecan-2 functions as a docking receptor for pro-MMP-7 in colon cancer cells.

Proteomic expression analysis of human colorectal cancer: Identification of soluble overexpressed proteins

Colorectal cancer is one of the most common malignancies in developed countries. Scarce clinical signs at the early stages of the disease and the lack of fast and sensitive diagnostic techniques based on the detection of tumor specific protein markers contribute greatly to the high mortality rate. The search for such markers is significantly complicated by the high levels of major structural and cytoskeletal proteins in normal and tumor tissues. Extraction with 0.2 M NaCl in the presence of the nonionic detergent NP-40 was performed to enrich the soluble protein fraction. This modification resulted in a considerably increased sensitivity of detection of minor proteins that may enter the circulation during carcinogenesis. The soluble protein profiles of the paired colon adenocarcinoma and normal tissue specimens were compared using 2D gel electrophoresis, which enabled the detection of 10 proteins whose levels in tumors were elevated at least 10-fold as compared to normal tissue. The proteins were identified by MALDI-TOF mass spectrometry, and two new protein markers of colon cancer, TAF9 and CISH, were discovered. Low levels of CISH synthesis in most normal human tissues and tumors other than colorectal cancer make it a prospective candidate diagnostic marker for this type of cancer.

Reduced hGC-1 Protein Expression Is Associated with Malignant Progression of Colon Carcinoma

hGC-1 (human granulocyte colony-stimulating factor-stimulated clone 1) is a gastrointestinal protein that is a member of the olfactomedin glycoprotein family. Its biological function remains poorly understood. Aberrant expression of hGC-1 in some human carcinomas has been recently reported. The purpose of this study was to examine hGC-1 expression in colon carcinoma and explore the relationship between hGC-1 expression and the clinicopathologic features of patients with colon cancer. The expression of hGC-1 in colon adenocarcinoma tissues was examined by dot-blot analysis, in situ hybridization, and immunohistochemistry. The association of hGC-1 expression pattern with patient differentiation grade, tumor stage, metastasis, and survival were examined. To further investigate the involvement of hGC-1 in colon cancer progression, human colon carcinoma (HT-29) cells overexpressing hGC-1 were established and cell proliferation, adhesion, and migration were studied. Compared with normal colon mucosa, the up-regulation of hGC-1 was more frequently detected in more differentiated colon cancers, whereas down-regulation or no expression was associated with poorly differentiated colon cancers. Interestingly, hGC-1 down-regulation was also found in late tumor-node-metastasis stage, metastasis, and in patients with shorter survival. The morphology and cortical actin distribution of HT-29 cells were altered by hGC-1 overexpression. However, this did not change cell proliferation, but decreased cell adhesion and migration. Our findings indicate that hGC-1 is involved in colon cancer adhesion and metastasis, and that hGC-1 may be a useful marker for tumor differentiation and progression of human colon carcinoma.

Novel fucogangliosides found in human colon adenocarcinoma tissues by means of glycomic analysis

The structures of acidic glycosphingolipids in colon adenocarcinoma have been analyzed extensively using a number of conventional methods, such as thin-layer chromatography and methylation analysis, and a variety of acidic glycosphingolipids present in the tissues have been reported. However, because of a number of limitations in the techniques used in previous studies in terms of resolution, quantification, and sensitivity, we employed a different method that could be applied to small amounts of tissue. In this technique, the carbohydrate moieties of acidic glycosphingolipids from approximately 20 mg of colon adenocarcinoma were released by endoglycoceramidase II and were labeled by pyridylamination. They were separated and structurally characterized by a two-dimensional HPLC mapping technique, electrospray ionization tandem mass spectrometry (ESI–MS/MS), and enzymatic cleavage. A total of 22 major acidic glycosphingolipid structures were identified, and their relative quantities were revealed in detail. They are composed of 1 sulfated (SM3), 1 lacto-series (SLe a), 6 kinds of ganglio-series, and 14 kinds of neolacto-series glycosphingolipids. They include most of the acidic glycosphingolipids previously reported to be present in the tissues and two previously unknown fucogangliosides sharing the same terminal structure: NeuAcα2-6(Fucα1-2)Galβ1-4GlcNAcβ1-3Galβ1-4Glc, and NeuAcα2-6(Fucα1-2)Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3-Galβ1-4Glc. Thus, this highly sensitive, high-resolution analysis enabled the identification of novel structures of acidic glycosphingolipids from small amounts of already comprehensively studied cancerous tissues. This method is a powerful tool for microanalysis of glycosphingolipid structures from small quantities of cancerous tissues and should be applicable to different types of malignant tissues.

IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1

Insulin-like growth factor binding-protein-7 (IGFBP7) was obtained from our previous colonic adenocarcinoma (CRC) and normal mucosa suppression subtraction hybridization (SSH) cDNA libraries. By RT-PCR and immunohistochemistry, we found that IGFBP7 was overexpressed in CRC tissue compared to normal tissue. However, our in vitro experiments performed in 10 CRC cell lines showed that IGFBP7 expressed only in SW480 and Caco2 cell lines, which implied an underlying reversible regulatory mechanism. Using methylation-specific PCR (MSP) and bisulfite sodium PCR (BSP), we found that its expression was associated with DNA hypomethylation of exon1. This was further supported by the in vitro study which showed restored IGFBP7 expression after demethylation agent 5-aza-2'-deoxycytidine treatment. Correlation analysis between IGFBP7 expression and prognosis indicated that overexpression of IGFBP7 in CRC tissue correlated with favourable survival. Investigation of the functional role of IGFBP7 through transfection studies showed that IGFBP7 protein could inhibit growth rate, decrease colony formation activity, and induce apoptosis in RKO and SW620 cells, suggesting it a potential tumor suppressor protein in colorectal carcinogenesis. In conclusion, our study clearly demonstrated that IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis and its expression is associated with DNA hypomethylation of exon 1.

Ectopic expression of N-acetylglucosamine 6-O-sulfotransferase 2 in chemotherapy-resistant ovarian adenocarcinomas

Mucinous and clear cell adenocarcinomas are the major histological types of ovarian epithelial cancer and are associated with a poor prognosis due to their resistance to chemotherapy. A novel tumor marker specific for ovarian mucinous and clear cell adenocarcinomas would be helpful for overcoming these serious diseases. We showed previously by enzymological characterization and RT-PCR that colonic mucinous adenocarcinoma tissues ectopically express GlcNAc6ST-2, a member of the carbohydrate 6-O-sulfotransferase family (Seko, A. et al. (2002) Glycobiology 12, 379-388). Here, we prepared a GlcNAc6ST-2-specific polyclonal antibody for immunohistochemical analysis and found that GlcNAc6ST-2 is ectopically expressed by not only colonic mucinous adenocarcinomas but also ovarian mucinous, clear cell and papillary serous adenocarcinomas. In contrast, solid serous adenocarcinomas, endometrioid adenocarcinomas, and mucinous adenomas expressed GlcNAc6ST-2 much less frequently or not at all. RT-PCR analysis confirmed that GlcNAc6ST-2 transcripts are expressed in ovarian mucinous adenocarcinomas but not in mucinous adenomas. In addition, immunohistochemical analysis using sulfated glycan-specific monoclonal antibodies showed that ovarian adenocarcinoma cells express GlcNAc 6-O-sulfated glycans, including an L-selectin ligand and its related glycans. These results indicate that GlcNAc6ST-2 would be a novel tumor antigen that is specifically expressed in ovarian mucinous, clear cell and papillary serous adenocarcinomas.

Physiological and Functional Interactions between Tcf4 and Daxx in Colon Cancer Cells

Daxx, a human cell death-associated protein, was isolated as a Tcf4-interacting protein, using a yeast two-hybrid screen. Co-immunoprecipitation in HEK-293T cells and yeast two-hybrid screen in Y190 cells were performed to identify the interaction between Tcf4 with Daxx and to map the binding regions of Tcf4. In the nucleus, Daxx reduced DNA binding activity of Tcf4 and repressed Tcf4 transcriptional activity. Overexpression of Daxx altered the expression of genes downstream of Tcf4, including cyclin D1 and Hath-1, and induced G1 phase arrest in colon cancer cells. A reduction in Daxx protein expression was also observed in colon adenocarcinoma tissue when compared with normal colon tissue. This evidence suggests a possible physiological function of Daxx, via interaction with Tcf4, to regulate proliferation and differentiation of colon cells.

Derivation and Characterization of a Monoclonal Antibody Against Human Glycinamide Ribonucleotide Formyltransferase

Glycinamide ribonucleotide formyltransferase (GARFT) is a trifunctional enzyme involved in purine biosynthesis. Its central role in folate metabolism has made it an obvious target for the development of GARFT inhibitors, primarily for oncology. While the crystal structure, enzyme kinetics, and mechanism of action of GARFT inhibitors are reasonably well understood, GARFT regulation at the protein level remains unclear. The present study reports the development and characterization of a monoclonal antibody (MAb) specific for human GARFT. This MAb, an IgG1kappa, designated PHR1, recognizes human GARFT by both Western blot and by immunohistochemistry from non-small-cell lung carcinoma and colon adenocarcinoma tissue biopsies, has a KD of 1.14 x 10(10) M, and has been epitope mapped at residues 59-78 of the GARFT functional domain. The ability of PHR1 to recognize both sodium dodecyl sulfate (SDS)-denatured as well as native GARFT should make this MAb an important research tool in determining GARFT protein levels in both normal and neoplastic tissues.

Functional expression of TWEAK in human colonic adenocarcinoma cells

The TNF-like weak inducer of apoptosis (TWEAK) can induce diverse cellular responses, including cell death, inflammation, migration, and proliferation in various transformed cell lines. We investigated TWEAK sensitivity, TWEAK effects on nuclear factor-kappaB activation, and expression of TWEAK in the HT-29, LS180, SK-CO-1 and SW480 human colonic adenocarcinoma cell lines, all of which express the TWEAK receptor (Fn14). TWEAK alone induced cell death in SW480 cells and induced cell death of HT-29 cells after addition of IFN-gamma, actinomycin D or cycloheximide. TWEAK did not affect cell viability of LS-180 or SK-CO-1 cells. Activation of NF-kappaB was not obviously influenced by TWEAK in any of the cell lines. All four human colonic adenocarcinoma cell lines constitutively expressed TWEAK mRNA, protein and membrane-bound TWEAK antigen, as detected by RT-PCR, Western blotting and flow cytometry. Stimulation by an anticancer drug (camptothecin) augmented cell surface expression of TWEAK and all human colonic adenocarcinoma tissue samples studied (n=59) demonstrated positive staining for TWEAK antigen. Soluble TWEAK was detected in culture medium of these cell lines by ELISA and conditioned medium from SW480 cells incubated with anti-TWEAK antibody significantly inhibited endothelial cell tube formation in Matrigels. Thus, functional expression of TWEAK from human colonic adenocarcinoma cells may contribute to neovascularization.

Overexpression of caveolin-1 in experimental colon adenocarcinomas and human colon cancer cell lines

Caveolin-1, -2, and -3 are the principal proteins of caveolae, the vesicular invaginations of the plasma membrane. Recent studies suggest that caveolins play an important role in cellular signaling and, possibly, in tumorigenesis. We examined the expression of the three caveolins in azoxymethane-induced rat colon adenocarcinoma and normal-appearing colonic mucosa, and also in human colon cancer cells with inherently different proliferation rates. Expression of caveolins was assessed in experimental rat colon adenocarcinoma and normal-appearing colonic tissues by RT-PCR, immunoblot and immunohistochemistry. Expression of caveolin-1, -2, and -3 at protein and mRNA levels in human colon cancer cell lines Caco-2, HT-29 and HCT-116 was assessed by immunoblot and RT-PCR, respectively. While caveolin-1 was overexpressed in all experimental adenocarcinoma tissues by comparison to normal-appearing colonic mucosa at both the mRNA and protein level, caveolin-2 was expressed equally only at the mRNA level in both tissues. Immunohistochemical analysis of experimental rat colon tissues also revealed a similar pattern with caveolin-1 being overexpressed in adenocarcinomas as compared to normal-appearing colonic mucosa. Caveolin-1 protein expression was observed in HT-29 and HCT-116 cell lines but not in the Caco-2 cell line or the macrophage Raw 264.7 cell line. Caveolin-1 mRNA expression was evident in all colon carcinoma cell lines tested; however, caveolin-2 mRNA was found only in HT-29 and HCT-116. Caveolin-1 protein and mRNA transcript levels in human colon cancer cell lines were observed to be concordant with the growth rate of the cells, and followed the pattern of HCT-116 > HT-29 > Caco-2. Taken together, our results indicate that caveolin-1 is overexpressed in experimental colon adenocarcinoma by comparison to adjacent normal mucosa, and its expression in human colon cancer cells is directly associated with the growth rate.

Biological functions of group X secretory PLA2.

Secretory phospholipase A2s (sPLA2s) are a diverse family of low molecular mass enzymes that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. In addition to the classical types of group IB and IIA (sPLA2-IB and IIA), recent advances in molecular biology have led to the identification of novel types of sPLA2s, including group IID and IIE that we have cloned [1]. Among the nine types of human sPLA2s, group X sPLA2 (sPLA2-X) has a structural similarity with the classical type of sPLA2-IB and IIA [2]. We have shown that sPLA2-X has a potent hydrolyzing activity toward phosphatidylcholine (PC) and elicits more potent production of unsaturated fatty acids and lysophosphatidylcholine (lysoPC) in various inflammatory cells and colon cancer cells compared with sPLA2-IB and IIA [3,4]. The released arachidonic acid by sPLA2-X was efficiently converted to prostaglandins (PGs) by the action of endogenous cyclooxygenase (COX). The expression of sPLA2-X was detected in human lung epithelial cells and mouse splenic macrophages, and greatly augmented in human colon adenocarcinoma tissues in concert with enhanced expression of COX-2 [4]. These findings suggest that sPLA2-X plays a critical role in the lipid mediator productions under various inflammatory states and in the COX-2- dependent PGE2 biosynthesis during colon tumorigenesis. Recently, we have shown that sPLA2-X is a high-affinity ligand for mouse PLA2 receptor (PLA2R) and identified a soluble PLA2R in mouse plasma [5]. Using PLA2 receptor-deficient mice, we have shown that the membrane and soluble form of the receptor are involved in the endogenous inhibitory mechanisms against the strong PC-hydrolizing activity of sPLA2-X in mice [5].

Analysis of molecular profile data using generative and discriminative methods.

A modular framework is proposed for modeling and understanding the relationships between molecular profile data and other domain knowledge using a combination of generative (here, graphical models) and discriminative [Support Vector Machines (SVMs)] methods. As illustration, naive Bayes models, simple graphical models, and SVMs were applied to published transcription profile data for 1,988 genes in 62 colon adenocarcinoma tissue specimens labeled as tumor or nontumor. These unsupervised and supervised learning methods identified three classes or subtypes of specimens, assigned tumor or nontumor labels to new specimens and detected six potentially mislabeled specimens. The probability parameters of the three classes were utilized to develop a novel gene relevance, ranking, and selection method. SVMs trained to discriminate nontumor from tumor specimens using only the 50-200 top-ranked genes had the same or better generalization performance than the full repertoire of 1,988 genes. Approximately 90 marker genes were pinpointed for use in understanding the basic biology of colon adenocarcinoma, defining targets for therapeutic intervention and developing diagnostic tools. These potential markers highlight the importance of tissue biology in the etiology of cancer. Comparative analysis of molecular profile data is proposed as a mechanism for predicting the physiological function of genes in instances when comparative sequence analysis proves uninformative, such as with human and yeast translationally controlled tumour protein. Graphical models and SVMs hold promise as the foundations for developing decision support systems for diagnosis, prognosis, and monitoring as well as inferring biological networks.

Down-regulation of drs mRNA in human colon adenocarcinomas.

We have previously reported that the drs gene, whose mRNA expression is down-regulated by retroviral oncogenes such as v-src and v-K-ras, has the ability to suppress transformation by v-src and v-K-ras in the rat cell line F2408. We have also isolated a human homolog of this gene (h-drs) and shown that expression of h-drs mRNA is markedly reduced in a variety of human cancer cell lines, including those of carcinomas of the colon, bladder, and ovary, suggesting that down-regulation of drs mRNA is correlated with the development of human cancers. To clarify the correlation between down-regulation of the drs gene and malignant tumor formation in human cancer tissues, we examined expression of drs mRNA in human normal tissues, colon adenoma, and adenocarcinoma tissues by in situ hybridization. Expression of drs mRNA was detected in most normal tissues tested, including those of the colon, bladder, and ovary. However, drs mRNA was hardly expressed in any of the colon adenocarcinoma tissues examined. Northern blot analyses confirmed these results. Neither gross deletion nor re-arrangement of the drs genome was detected by Southern blot hybridization in these adenocarcinoma tissues. drs mRNA was significantly expressed in colon adenoma with mild atypia but down-regulated in adenomas with moderate atypia and focal carcinoma. Our results indicate that down-regulation of drs mRNA is closely correlated with development of colon adenocarcinoma, suggesting a tumor-suppressor function of the drs gene in human cancers.

CD4- and TCRalphabeta-positive T lymphocytes predominantly infiltrated into well-moderately differentiated colon adenocarcinoma tissues.

Intraepithelial T lymphocytes have been reported as being functional in the growth of epithelial cells and also in the discrimination of aberrant cells, whereas their function against colon adenocarcinoma cells is obscure. The phenotype of colon intraepithelial T lymphocytes has been found in patients with inflammatory bowel diseases but not in patients with colon adenocarcinoma. In this study, we investigated cell surface markers of tumor-infiltrating T lymphocytes of adenocarcinoma of the colon and rectum at various grades of differentiation and intraepithelial T lymphocytes of adjacent normal colon by enzyme immunostaining. Among intraepithelial T lymphocytes of the normal colon, CD8-and TCRgammadelta-positive T lymphocytes were predominant as described. In contrast, tumor-infiltrating T lymphocytes of well-moderately differentiated adenocarcinoma of the colon and rectum were predominantly CD4- and TCRalphabeta-positive. The decrease of TCRgammadelta-positive T lymphocytes and the increase of CD4 and TCRalphabeta-positive T lymphocytes in adenocarcinoma tissues of the colon and rectum suggests that an alteration of the local immune system participates in the formation of adenoma and/or adenocarcinoma of the colon and rectum, resulting in infiltration of CD4-positive T lymphocytes that have certain activity against transformed cells.