Functional expression of tumor necrosis factor-related apoptosis-inducing ligand in human colonic adenocarcinoma cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. Therefore, we investigated TRAIL sensitivity, TRAIL-induced nuclear factor-kappaB (NF-kappaB) activation, and expression of TRAIL in human colonic adenocarcinoma cell lines (HT-29, LS180, SK-CO-1). All four TRAIL receptors (TRAIL-R1 through TRAIL-R4) are expressed in these cell lines. TRAIL sensitivity was assessed by assay of cell viability. Cancer cell viabilities were 83 +/- 3.1% (HT-29), 90 +/- 4.3% (LS180), and 88 +/- 6.3% (SK-CO-1) at 24 hours after the addition of 100 ng/ml TRAIL, indicating that these cell lines were relatively resistant to TRAIL. Activation of NF-kappaB was variably influenced by TRAIL administration, with no consistent tendency among the cell lines, indicating that TRAIL-induced NF-kappaB activation might be cell-type dependent. In contrast, TRAIL was expressed in the human colonic adenocarcinoma cell lines by Western blotting and RT-PCR. Increased expression of TRAIL on tumor cells was observed by flow cytometry after cytokine stimulation (IFN-gamma, TNF-alpha) or the addition of chemotherapeutic agents (camptothecin, doxolubicin hydrochloride). TRAIL on HT-29 cells was functional and able to induce apoptosis in Jurkat cells. Jurkat cell viability was increased by the addition of TRAILR1-R4-Fc. In the presence of various cytokines or chemotherapeutic agents, functional TRAIL is expressed on the surface of tumor cells, and this expressed TRAIL might contribute to tumor immune privilege by inducing apoptosis of activated human lymphocytes.     

Epimorphin expression in human colonic myofibroblasts

Epimorphin is a membrane-associated protein that has been postulated to regulate epithelial morphogenesis in several tissues. However, epimorphin expression in the human intestine has not been fully investigated. In this study, we investigated epimorphin expression in the inflamed mucosa of inflammatory bowel disease (IBD). Tissue samples were obtained surgically from patients with active ulcerative colitis (UC) (n=5) and active Crohn's disease (CD) (n=5). Epimorphin and alpha-smooth muscle actin (SMA) were stained immunohistochemically. Epimorphin expression in human intestinal subepithelial myofibroblasts (SEMFs) was analyzed by Western and Northern blotting. In the normal colon, epimorphin expression was detected partly in the alpha-SMA-positive cells under the epithelial cells. Epimorphin was also expressed in alpha-SMA-positive cells in the capillary wall. In the inflamed mucosa of UC and CD patients, epimorphin expression was not altered. In isolated human SEMFs, epimorphin was detected as a single band of molecular weight 34-kDa under reducing and non-reducing conditions. In intestinal SEMFs, epimorphin mRNA expression was not affected by inflammatory cytokines and growth factors. Epimorphin was constitutively expressed in the normal colonic mucosa, and this was not altered in the inflamed mucosa of IBD patients. The localization of epimorphin may indicate a potential role in maintaining normal tissue structure in normal and IBD mucosa.     

Tetranectin expression in human colonic neoplasia

Pigmented lesions of palmar and plantar skin may cause diagnostic problems, partly because they are infrequently excised and also because some features of benign lesions in these sites may raise the suspicion of melanoma if considered alone. We have examined a series of benign melanocytic lesions and compared them with melanomas from these sites. The presence of severe melanocytic atypia was the most valuable feature in distinguishing between naevi and melanomas. Pagetoid infiltration of the epidermis by single atypical cells, or small groups of cells with abundant pale cytoplasm was seen only in melanomas, while transepidermal elimination of well-circumscribed nests was present only in benign lesions. A lymphocytic infiltrate was present in the dermis in 13 of 14 malignant lesions, but only two of the 26 naevi showed a sparse infiltrate: we suggest that the presence of a lymphocytic infiltrate should prompt a careful search for other features of malignancy. Other features examined, including elongation of rete ridges, pattern of melanocyte distribution at the dermo-epidermal junction, dermal sclerosis, and pigment in the stratum corneum or in the dermis, were seen in both naevi and melanomas and were not found to be useful in distinguishing benign from malignant lesions.     

HLA-DR expression in a human colonic carcinoma cell line

A human colonic carcinoma cell line (HCA-7) isolated from a well differentiated mucoid adenocarcinoma of the colon has been maintained in vitro for 3 years. It spontaneously synthesizes HLA-DR which is mainly intracytoplasmic. Stimulation with lymphocyte conditioned medium and recombinant gamma-interferon results in enhanced synthesis of HLA-DR and the appearance of the antigen on the cell surface. A dose response study showed that maximal stimulation of the culture was achieved with 50 units/ml of recombinant gamma-interferon. Staining for HLA-DR was uneven being confined to focal areas of the monolayer, which is similar to the focal expression of HLA-DR seen in sections of adenocarcinoma of the colon. The functional significance of this phenomenon is unclear, but it may explain the presence of lymphoid infiltrates in tumours.     

Chromogranin A is a T cell antigen in human type 1 diabetes

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.     

Conversion of human colonic adenoma cells to adenocarcinoma cells through inflammation in nude mice

The roles of inflammation in the malignant progression of tumors during multistep carcinogenesis have been much discussed but remain to be elucidated. To determine the direct contribution of inflammation to colon carcinogenesis, we established a new model of progression of human colonic adenoma cells using a nude mouse; the progression is accelerated by coimplantation of a plastic plate. The FPCK-1-1 cell line, derived from a colonic polyp in a patient with familial adenomatous polyposis, is nontumorigenic when injected subcutaneously into nude mice in a cell suspension of up to 5 x 106 cells per mouse. However implantation of 1 x 10(5) FPCK-1-1 cells attached to a plastic plate induced first acute and then chronic inflammation, and formed progressively growing tumors that were histologically determined as moderately differentiated adenocarcinoma in 65% of mice. Moreover cell lines established from the growing tumors were found to be tumorigenic when injected into mice even without a plastic plate. The tumor arising from the adenoma cells implanted attached to a plastic plate was surrounded by highly proliferating fibrous stroma. This fibrous tissue was considered essential for malignant progression, rather than for attachment to the plastic plate substrate, because the tumors were formed after injection of FPCK-1-1 cells into the fibrous tissue from which the plastic plate had been removed before the cell injection. The conditioned medium (CM) obtained from the fibroblasts derived from a plastic plate-associated stromal tissue was found to contain factors that stimulated growth of FPCK-1-1 cells, but not of the derivative progressor cell lines. The factor was stable to heating and neuraminidase treatment, but labile to trypsin treatment. The main growth-potentiating activity was contained in the fraction larger than 100 kDa. In contrast, the activity to promote FPCK-1-1 cell growth was not present in the CM of subcutaneous fibroblasts from untreated nude mice or the fibroblast cell lines C3H10T 1/2 and NIH3T3. These results demonstrated that inflammation-associated stroma promoted the conversion of colonic adenoma cells to adenocarcinoma cells.     

Loss of Reprimo and S100A2 expression in human gastric adenocarcinoma

Reprimo and S100A2 are two newly identified candidate tumor-suppressor genes, which play an important role in the regulation of p53-dependent cell cycle. In this study, we examined the expressions of Reprimo and S100A2 in surgical specimens of gastric adenocarcinoma and correlated these results with pathological and clinical parameters. Tissues were obtained from 100 gastric adenocarcinoma patients that underwent curative gastrectomy. Reprimo and S100A2 expressions were evaluated by immunohistochemical analysis. Loss of Reprimo and S100A2 expressions occurred in 65 and 52% of the patients, respectively. Loss of Reprimo expression was significantly correlated with the depth of tumor invasion (P = 0.000), lymphatic vessel invasion (P = 0.006), and lymph node metastasis (P = 0.000). Loss of S100A2 expression was significantly associated with histological type (P = 0.009), depth of invasion (P = 0.033), lymphatic vessel invasion (P = 0.01), and lymph node metastasis (P = 0.001). In addition, there was a significant positive association between the expressions of Reprimo and S100A2 (P < 0.01). The results suggest that loss of Reprimo and S100A2 expressions occurs frequently in gastric adenocarcinomas. The expressions of Reprimo and S100A2 may be potential biomarkers for gastric adenocarcinomas detection.     

Regulation of amphiregulin and epiregulin expression in human colonic subepithelial myofibroblasts

Amphiregulin and epiregulin belong to the epidermal growth factor (EGF) family, and act as mitogenic stimulators via binding to EGF receptors (EGFRs). Amphiregulin and epiregulin are thought to play a role in regenerative responses in the gastrointestinal tract. In this study, we investigated secretion of amphiregulin and epiregulin in human colonic subepithelial myofibroblasts (SEMFs). The mRNA expression and protein secretion of amphiregulin and epiregulin were evaluated by Northern blotting and Western blotting, respectively. The trophic effects of amphiregulin and epiregulin on SEMFs were analyzed by MTT assays. Amphiregulin and epiregulin mRNAs were not detected in unstimulated SEMFs. Among the various cytokines and growth factors, interleukin-1beta, tumor necrosis factor-alpha, and EGF strongly induced amphiregulin and epiregulin mRNA expression. These responses were markedly reduced by AG1478, a specific inhibitor of EGF receptor tyrosine kinases. Amphiregulin and epiregulin secretion were also detected at the protein level. MTT assays demonstrated that amphiregulin and epiregulin stimulate the proliferation of SEMFs. We demonstrated expression of amphiregulin and epiregulin in SEMFs. Amphiregulin and epiregulin may play an important role in the mechanism underlying wound healing in damaged colonic mucosa.     

Chromogranin A, B, and C in human adrenal medulla and endocrine tissues

Antisera against bovine chromogranin A, B, and C were used to identify these proteins in human tissues by one- and two-dimensional immunoblotting and by immunohistochemistry. Human chromaffin granules contain all three chromogranins. Their molecular weight differs slightly from the bovine proteins. Chromogranin B and to a lesser degree chromogranin A are apparently processed by endogenous proteases yielding several smaller proteins which cross-react immunologically. In human granules the two families of chromogranins A and B represent the major protein groups, whereas in bovine granules the chromogranins A are by far the most prominent component. All three chromogranins are also found in the anterior pituitary. In immunoblotting these pituitary proteins behave identically to the adrenal chromogranins. In immunohistochemistry a portion of the pituitary cells stains for all three chromogranins. In endocrine pancreas only chromogranin A and B could be found, whereas in the parathyroid gland only chromogranin A is present. This study establishes that human chromaffin granules contain three immunologically distinct groups of acidic proteins. These chromogranins are also found in other endocrine tissues but they are not always stored together. Antisera against these three proteins might be used as immunological markers for tumors derived from endocrine tissue.     

KLF15在人肺腺癌中表达的临床意义

目的:Krüppel样因子15(Krüppel-like factor 15,KLF15)参与肿瘤的增殖、侵袭和转移,但在肺腺癌中的表达和作用尚不明确。本研究探索KLF15蛋白在肺腺癌患者中的表达及其临床意义。方法:收集我院4例肺腺癌组织及匹配的癌旁组织,Western blot分析这些组织中KLF15蛋白表达的差异。同时收集我院72例肺腺癌患者的肿瘤组织标本及临床资料,免疫组织化学染色分析患者肺癌标本中KLF15蛋白的表达,分析其表达与患者临床特征包括预后的关系。另外,采用质粒转染的方法上调肺腺癌A549细胞中KLF15的表达,CCK-8法分析KLF15蛋白对肺腺癌细胞增殖能力的影响。结果:在4例肺腺癌及匹配的癌旁组织中,肺腺癌组织中KLF15蛋白表明显低于癌旁组织。在72例肺腺癌病理标本中,KLF15蛋白低表达或不表达者53例,占73.6%,KLF15高表达者19例,占26.4%。KLF15蛋白高表达的患者,其5年生存率明显优于KLF15低表达的患者(P0.01);KLF15蛋白表达与肺腺癌患者的病理分期(P0.01)及T分期明显相关(P0.01),KLF15蛋白低表达是肺腺癌患者不良预后的重要危险因素。上调肺腺癌细胞中KLF15蛋白的表达可明显抑制肺腺癌细胞的生长。结论:KLF15抑制肺腺癌细胞的生长,有望成为肺腺癌患者的治疗靶点和预后分子标志物。