Expression of Frizzled5, Frizzled7, and Frizzled10 during early mouse development and interactions with canonical Wnt signaling.

Wnt signaling has been shown to be important in the patterning of the gastrulating mouse embryo, especially in axis formation. To this date, there is no clear indication that the Wnt receptors, Frizzleds (Fzds), are involved in such early specification. Moreover, at the gastrulation stage, the only Fzd with a known characterized expression pattern is Fzd8, which is expressed in the anterior visceral endoderm (aVE) (Lu et al. [2004] Gene Expr Patterns 4:569-572). Following a real time RT-PCR study to evaluate Fzd expression in the gastrulating embryo, we used whole-mount in situ hybridization to reveal new expression domains for Fzd5, Fzd7, and Fzd10. Fzd5 is expressed in the aVE and Fzd7 expression is restricted to the epiblast of the gastrulating embryo. The expression pattern of Fzd10 in the primitive streak of the gastrula suggests it has a role in mesoderm induction. We also show that the purified, secreted forms of the extracellular cysteine-rich domains (CRDs) of FZD5, Fzd7, and Fzd8 can antagonize Wnt3a-induced beta-Catenin accumulation in L-cells, whereas in mouse embryonic stem cells, these CRDs can inhibit spontaneous mesoderm formation and promote neural differentiation. Our data demonstrate that Fzd5, Fzd7, and Fzd10 are expressed in distinct domains of the gastrulating embryo, and that the CRDs of FZD5, Fzd7, and Fzd8 can regulate Wnts, indicating that Fzds interpret Wnt signals during embryonic mesoderm and neural induction.     

Quantification of pancreatic secretory trypsin inhibitor in colonic carcinoma and normal adjacent colonic mucosa.

AIMS: To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. METHODS: From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dose-dependent manner. The specimens were obtained from resectional surgery. RESULTS: The content of irPSTI was 19.9 micrograms/g protein (0.55 micrograms/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43.6 micrograms/g protein (1.12 micrograms/g tissue wet weight) was shown. CONCLUSIONS: The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma.     

Expression of Wnt, Frizzled, sFRP, and DKK genes in adult human pancreas

Wnts are important signaling molecules involved in many normal developmental processes in the human body as well as some forms of cancer. Nineteen Wnt genes are found in the human genome, as well as 10 Wnt receptor genes called Frizzled. Two coreceptors called LRP 5 and 6 are critical for Wnt signal transduction. The interaction of the Wnts with the receptors is regulated by two classes of extracellular Wnt or LRP binding proteins called sFRP and Dickkopf (DKK), which modulate Wnt signaling. We have examined the expression of all Wnt family members both in the exocrine portion and in isolated islets of adult human pancreas. RT-PCR analysis of the 1-day cultured exocrine pellet fraction from the islet isolation procedure showed that Wnt 2, 2b, 3, 4, 5a, 5b, 7a, 7b, 14, and 15 were detectable. All 10 Frizzled (Frz) receptors were expressed but only Frizzled 1, 2, 4, 5, and 6 strongly. RT-PCR performed on purified human islets revealed that Wnt 2b, 3, 4, 5a, 7b, 10a, and 14 and Frz 4, 5, and 6 were the most highly expressed. DKK 1, 3, and 4 as well as sFRP 1, 4, and 5 were expressed in the exocrine fraction. sFRP 2 and 3 were detectable but only at low levels. In situ hybridization for Frz 1-7 showed that expression colocalized with the islets of Langerhans. Together the data suggest that active Wnt signaling occurs in adult pancreas and is probably important for physiological functions.     

Induction of angiogenesis by hyperplastic colonic mucosa adjacent to colon cancer

We determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-alpha than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-beta inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-beta than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis.     

Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas, and carcinomas

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cell lines. Four membrane-bound receptors for TRAIL have been identified, two apoptosis-mediating receptors, DR4 and DR5, and two apoptosis-inhibiting receptors, DcR1 and DcR2. The aim of this study was to examine the role of TRAIL and its receptors in colorectal cancer development. The immunohistochemical expression and localization of TRAIL and its receptors were investigated in normal mucosa (n=10), adenomas (n=19), and carcinomas (n=21). Correlations between the expression of TRAIL and its receptors and the degree of apoptosis (assessed by M30 expression) and histopathological characteristics were explored. TRAIL and its receptors were expressed in normal mucosal epithelium. Expression of the receptors was seen in adenomas and carcinomas. TRAIL expression was lost in a subset of colorectal tumours, more frequently in carcinomas than in adenomas (p<0.05). DR4 and DR5 staining was stronger in neoplastic cells than in normal cells and was accompanied by a higher degree of apoptosis. No differences were found between tumour and normal cells regarding DcR1 and DcR2 expression. No correlations were found between TRAIL or TRAIL receptor expression and histopathological characteristics. In conclusion, marked changes were seen in the course of the adenoma-carcinoma sequence with respect to the expression of TRAIL and TRAIL receptors DR4 and DR5. The stronger expression of DR4 and DR5 in neoplastic cells than in normal cells, together with a higher degree of apoptosis, suggests a possible functional role for these receptors in apoptosis induction in neoplastic colorectal cells.     

Expression of survivin in normal, hyperplastic, and neoplastic colonic mucosa

The regulation of apoptotic cell death may have a profound effect on the pathogenesis and progression of colon cancer. Survivin, a member of the inhibitor of apoptosis gene family, has been detected in fetal tissue and in a variety of human malignancies. In the current study, we investigated survivin expression by an immunohistochemical approach in benign, hyperplastic, premalignant, and malignant lesions of the colon. Survivin was detected in all cases of normal colonic mucosa (20/20), hyperplastic polyps (20/20), adenomatous polyps (20/20), and in both well differentiated and moderately differentiated colonic adenocarcinomas (20/20). In the normal colonic mucosa, survivin expression was mostly restricted to the base of the colonic crypts. All epithelial cells showed uniformly intense staining for survivin in hyperplastic polyps. By contrast, adenomas and adenocarcinomas showed a heterogeneous staining pattern with cell-to-cell, gland-to-gland, and regional variability in the intensity of survivin staining. In contrast to the basal preponderance of staining in normal colonic mucosa, numerous survivin positive cells were present at the luminal surface of hyperplastic polyps, adenomatous polyps, and adenocarcinomas. In conclusion, the expression of survivin is not a specific marker of adenocarcinoma of the colon but does show characteristic and reproducible patterns of expression in non-neoplastic proliferative lesions and in normal colonic mucosa.     

Expression of heparanase in normal, dysplastic, and neoplastic human colonic mucosa and stroma. Evidence for its role in colonic tumorigenesis

The human heparanase gene, an endo-beta-glucuronidase that cleaves heparan sulfate at specific intrachain sites, has recently been cloned and shown to function in tumor progression and metastatic spread. Antisense digoxigenin-labeled heparanase RNA probe and monoclonal anti-human heparanase antibodies were used to examine the expression of the heparanase gene and protein in normal, dysplastic, and neoplastic human colonic mucosa. To our knowledge, this is the first systematic study of heparanase expression in human colon cancer. Both the heparanase gene and protein were expressed at early stages of neoplasia, already at the stage of adenoma, but were practically not detected in the adjacent normal-looking colon epithelium. Gradually increasing expression of heparanase was evident as the cells progressed from severe dysplasia through well-differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells showed the highest levels of the heparanase mRNA and protein associated with expression of both the gene and enzyme by adjacent desmoplastic stromal fibroblasts. A high expression was also found in colon carcinoma metastases to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts. Moreover, extracts derived from tumor tissue expressed much higher levels of the heparanase protein and activity as compared to the normal colon tissue. In all specimens, the heparanase gene and protein exhibited the same pattern of expression. These results suggest a role of heparanase in colon cancer progression and may have both prognostic and therapeutic applications.     

酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及其功能

目的 探讨酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及作用。 方法 应用Western blotting和RT-PCR技术检测c-kit基因及KIT
蛋白在野生型C57BL/6小鼠结肠黏膜上皮的表达;用免疫荧光染色显示野生型C57BL/6小鼠以及乙基亚硝酸钠（ENU）诱变的c-kit基因点突变纯
合子小鼠（Wads ^m/m）结肠黏膜上皮KIT阳性细胞的部位;腹腔注射5-溴脱氧尿嘧啶核苷（BrdU）（30 mg/kg）观察对比野生型以及Wads m/m
小鼠结肠黏膜上皮BrdU掺入情况及动态变化,分析KIT在结肠黏膜上皮更新过程的作用。 结果 RT-PCR检测结果表明,在野生型小鼠结肠黏膜组
织表达 c-kit基因, Western blotting证明在肠黏膜组织存在KIT蛋白;免疫荧光染色显示,KIT阳性细胞位于结肠黏膜上皮,主要位于肠腺隐
窝基底部,但是Wads ^m/m小鼠KIT阳性细胞数以及KIT蛋白表达量较野生型小鼠明显减少;BrdU掺入实验发现,Wads ^m/m小鼠远端结肠黏膜上皮
BrdU摄入和更新速度较野生型小鼠明显减慢。结论 结肠黏膜上皮表达KIT蛋白与结肠黏膜上皮的更新密切相关。     

结直肠癌及结直肠粘膜p16基因5‘CpG岛甲基化状态的研究

目的 了解国人结直肠癌及正常肠粘膜Ｐ１６基因５＇ＣｐＧ岛甲基化状况,并探讨其意义。方法 用甲基化敏感的限制笥内切酶消化结合ＰＣＲ法检测６０例结直肠癌及其正常结直肠粘膜Ｐ１６基因５＇ＣＰＧ岛中ＨｐａⅡ、ＳａｃⅡ、Ｓｍａ位 甲基化状况。结果 结直肠癌与正常结直肠粘膜的Ｐ１６基因５＇ＣＰＧ岛均有不同程度的甲基化,比较结直肠癌与正常结直肠粘膜的Ｐ１６基因甲基化状态及其与临床病理资料的关系,差异均无显性。     

Expression of c-erbB receptors and ligands in the bronchial epithelium of asthmatic subjects.

BACKGROUND: The c-erbB family of receptor tyrosine kinases act in a combinatorial fashion to regulate cell behavior. Disturbances in this system have been associated with neoplastic and inflammatory diseases. OBJECTIVES: Although expression of the epidermal growth factor receptor (EGFR; c-erbB1) is increased in the bronchial epithelium in asthma, there is no information on expression of other members of the c-erbB receptor and ligand family that can modulate EGFR function. METHODS: Immunohistochemistry was used to compare expression of EGFR, c-erbB2, c-erbB3, epidermal growth factor, heparin-binding epidermal growth factor-like growth factor, and transforming growth factor alpha in bronchial biopsy specimens from normal and asthmatic subjects. Scrape-wounded monolayers of 16HBE 14o(-) cells were used as an in vitro model of damage and repair. Changes in EGFR, c-erbB2, and c-erbB3 distribution were measured by means of immunocytochemistry, whereas tyrosine phosphorylation was measured by means of immunoprecipitation and Western blotting. RESULTS: Although epithelial staining for the EGFR was significantly increased in asthmatic epithelium (P <.001), there was no difference in staining for the other receptors and ligands studied. In scrape-wounded epithelial monolayers, tyrosine phosphorylation of EGFR, c-erbB2, and c-erbB3 occurred immediately after damage; however, only EGFR showed a change in expression in response to damage. CONCLUSIONS: Even though EGFR levels are increased in asthma, this is not linked to changes in expression of its activating ligands or other c-erbB receptors. Because bronchial epithelial cells respond to physical damage through activation of several c-erbB family members, the shift in favor of increased EGFR levels in asthma may lead to altered epithelial function by influencing the number and type of heterodimeric signaling complexes, assuming sufficient ligand availability.     

结直肠癌及结直肠粘膜p16基因5′CpG岛甲基化状态的研究

目的　了解国人结直肠癌及正常肠粘膜p16基因 5′CpG岛甲基化状况 ,并探讨其意义。方法　用甲基化敏感的限制性内切酶消化结合PCR法检测 6 0例结直肠癌及其正常结直肠粘膜p16基因 5′CpG岛中HpaⅡ、SacⅡ、SmaⅠ位点的甲基化状况。结果　结直肠癌与正常结直肠粘膜的p16基因 5′CpG岛均有不同程度的甲基化 ,比较结直肠癌与正常结直肠粘膜的p16基因甲基化状态及其与临床病理资料的关系 ,差异均无显著性。正常结直肠粘膜的p16基因甲基化状态在不同的酶切位点存在差异 (P <0 .0 5 )。结直肠粘膜甲基化与年龄有相关趋势。结论　结直肠粘膜的p16基因甲基化在不同的酶切位点间存在差异。正常结直肠粘膜和结直肠癌的p16基因甲基化状态是比较复杂的。结直肠粘膜甲基化率有随年龄增加而增高趋势     

Expression of selectin ligands by cutaneous squamous cell carcinoma.

Recent evidence suggests that interactions between endothelial selectins and tumor surface selectin ligands may be of importance in cancer metastasis. To investigate the role of such mechanisms in cutaneous tumors, whole skin biopsies were examined immunohistochemically for a variety of selectin ligands including sialyl-Lewis-X, sialyl-Lewis-A (S-Le(a)), sulfatides, and CD15. In 12 of 12 squamous cell carcinomas (SCCs), there was expression of sialyl-Lewis-X and CD15, but no tumor expressed S-Le(a). Occasional keratinocytes in eight of 12 SCCs expressed sulfatides. All selectin ligands were absent on keratinocytes in basal cell carcinomas (BCCs, n = 8) and normal skin (n = 8), with the exception of one BCC that expressed S-Le(a). E-selectin was not present in normal skin, but was strongly expressed by dermal endothelium in both SCC and BCC. Keratinocyte cell lines A431, HaCaT, and SVK14 were investigated by flow cytometry, which demonstrated sialyl-Lewis-X and S-Le(a) expression by all three, whereas normal human keratinocytes did not express these molecules. These findings suggest a potential role for selectin-mediated events in early and late metastasis, and differential expression of these ligands by BCC and SCC may explain the relatively low metastatic potential of the former.     

大肠癌旁移行粘膜的增殖活性及其分布

目的：为进一步认识大肠癌“移行粘膜”（ＴＭ）的性质及为手术切除范围提供病理学依据。方法：通过连续观察２５例大肠癌癌旁１０ｃｍ范围内粘膜上皮细胞增殖活性的变化与正常结肠粘膜进行比较，发现ＴＭ在癌旁两边６ｃｍ范围内和近切端多春唾液酸粘蛋白反应、ＡｇＮＯＲｓ核均数、ＰＣＮＡ 率、Ｐ５３蛋白阳性等增殖活性均介于癌和正常结肠粘膜之间。结论：ＴＭ是腺体粘蛋白合成异常、增殖活性较高的一种癌前病变。手术切除肠段地     

Mononuclear cells in normal colon and colonic carcinoma express the same T cell activation markers.

The proportion of mononuclear cells (MC) expressing some of the T cell activation markers in normal colon and colonic carcinomas was determined by immunoperoxidase staining and computer-assisted video image analysis (VIA). Tissue sections were stained with monoclonal antibodies against the T cell activation markers which included the MHC class II antigens DR, DP and DQ, interleukin 2 receptor (CD25) and the p180 (CD45RO) form of the leucocyte common antigen. The mean values for the proportion of leucocytes expressing these activation markers were 92% for DR, 61% for DP, 68% for DQ and 60% for CD45RO in tumours and 90, 62, 70 and 55% in normal tissue respectively. Few cells were stained for the IL2 receptor (mean values 7% for tumours and 5% for normal tissue) or the p220 form (CD45RA) of the leucocyte common antigen (mean values 6% for tumours and 4% for normal tissue). The proportion of MC bearing any of these markers in the normal colon was not significantly different from the matched colonic carcinomas.