Is stromal microinvasion in primary mucinous ovarian tumors with "mucin granuloma" true invasion?

Stromal microinvasion has been recently described in ovarian mucinous borderline tumors (MBTs), leading to proposals for new classifications, including a category of MBTs with stromal microinvasion. This study was conducted to test the validity of this belief. To determine whether stromal microinvasion can be distinguishable from extruded neoplastic epithelium from an adjacent ruptured gland, particularly if accompanied by a mucin granuloma (MG) on hematoxylin and eosin (H&E)-stained sections, we compared the histopathologic features of 138 primary ovarian mucinous tumors, consisting of 81 MBTs, 37 MBTs with stromal microinvasion, 11 intraglandular carcinomas, 2 with microinvasive foci, and 7 mucinous adenocarcinomas with extensive stromal invasion. Immunohistochemical analysis for cytokeratin was performed in 72 cases. Of 77 cases containing MGs, 52% contained "microinvasive foci," consisting of individually scattered epithelial or irregular glandular components in the intervening stroma, as determined by H&E staining. Of the 37 negative cases on H&E, cytokeratin reactivity was detected in 15 additional cases with epithelial/glandular components. Therefore, "stromal microinvasion" can be missed on H&E. All patients with MBT with or without microinvasive foci and localized intraglandular carcinoma with or without microinvasive foci had stage I tumors; none died of tumor-related causes during the follow-up period. Tumor-related deaths were identified only in 5 patients with extensive stromal invasion. This study suggests that some cases of stromal microinvasion in stage I MBTs not accompanied by high grade nuclear atypia represent mucocele-like stromal reaction to ruptured mucinous glands rather than true stromal invasion because isolated tumor cells were exclusively confined to the boundary of MGs and devoid of obvious cellular atypia, and no patient with stromal microinvasion died of tumor-related causes.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

TC-1 (c8orf4) enhances aggressive biologic behavior in lung cancer through the Wnt/β-catenin pathway

Background

The thyroid cancer-1 (TC-1) or c8orf4 gene encodes a 106-residue naturally disordered protein that has been found to be associated with thyroid, gastric, and breast cancer. A recent study has indicated that the protein functions as a positive regulator in the Wnt/β-catenin signaling pathway in human breast cancer. However, no research has been done in the area of lung cancer. Therefore, the goal of the present study was to confirm the relationship among TC-1, lung cancer, and the Wnt/β-catenin signaling pathway.

Materials and methods

The expression of TC-1 was immunohistochemically examined in 147 patients with non–small-cell lung cancer. TC-1–overexpressed and silenced A549 cells were infected using lentivirus and MTT cell proliferation analysis, and Matrigel invasion assays and scratch-wound assays were performed to confirm the biologic behavioral changes in different A549 cell subsets. The Wnt/β-catenin signaling pathway, key gene β-catenin, target genes of vascular endothelial growth factor, cyclin D1, matrix metalloproteinase-7, c-myc, and survivin were tested at the mRNA and protein level.

Results

TC-1 was detected in 97 of the 147 non–small-cell lung cancer primary tumor specimens, and its expression correlated with the TNM stage and regional lymph node metastasis (P < 0.01). In vitro experiments demonstrated that TC-1 expression affected both proliferation and invasion in the A549 cell line. Furthermore, expression of TC-1 protein affected the Wnt/β-catenin signaling pathway’s downstream genes, such as vascular endothelial growth factor and matrix metalloproteinase-7, at the mRNA and protein level.

Conclusions

TC-1 expression is associated with aggressive biologic behavior in lung cancer and might coordinate with the Wnt/β-catenin pathway as a positive upstream regulator that induces these behaviors.     

β-catenin基因突变及表达与肝癌的关系(文献综述)

β-连环素 (β- catenin)是由 CTNNB1基因编码的一种多功能蛋白质 ,具有介导细胞间粘附及信号转导两大功能。近来的研究发现其编码基因在多种肿瘤中存在突变 ,对肝癌的研究表明 β- catenin基因突变和蛋白的过表达与肿瘤的发生、发展及预后密切相关。阻断 β- catenin信号通路的异常激活可能是治疗肝癌的一种新途径。     

Simvastatin induces estrogen receptor-alpha (ER-α) in murine bone marrow stromal cells

Simvastatin has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which simvastatin exerts its effects is still unclear. We previously reported that simvastatin promotes bone morphogenetic protein 2 (BMP-2) expression, induces osteoblastic differentiation, and inhibits adipocytic differentiation in mouse bone marrow stromal cells (BMSCs), and that this occurs, at least in part, via a BMP-2-dependent pathway. The aim of this study was to investigate further the mechanisms by which simvastatin stimulates osteogenesis in mouse BMSCs. To determine whether simvastatin-mediated osteogenesis was dependent on BMP-2, mouse BMSCs were treated with nonimmune normal mouse IgG or BMP-2 neutralizing antibodies combined with different concentrations of simvastatin. Surprisingly, the stimulatory effect of simvastatin on alkaline phosphatase (ALP) activity was not completely blocked by neutralizing BMP-2 monoclonal antibody treatment. Interestingly, we found that estrogen receptor-alpha (ER-α) protein levels increased after mouse BMSCs were treated with simvastatin for 72 h in a concentration-dependent manner. Moreover, the stimulatory effect of simvastatin on ALP activity in BMSCs was blocked by the estrogen receptor agonist ICI 182,780, and cotreatment with 17-β-estradiol and simvastatin increased ALP activities by two-to threefold in the BMSCs compared with treatment with simvastatin alone. These results suggest that simvastatin-induced in vitro osteogenesis in mouse BMSCs is mediated, at least in part, by induction of ER-α and not by BMP-2 alone. These results provide new insight into the mechanisms of simvastatin-induced bone formation in BMSCs.     

Elevated transforming growth factor-beta 1 (TGF-β1) levels in human fracture healing

Introduction

Transforming growth factor-beta 1(TGF-β1) is a regulatory protein, involved in bone fracture healing. Circulating TGF-β1 levels have been reported to be a predictor of delayed bone healing and non-union, suggesting active relationship between tissue and circulating TGF-β1 in fracture healing. The purpose of this study was to analyse TGF-β1 local and serum concentrations in fracture healing to further contribute to the understanding of molecular regulation of fracture healing.

Patients and methods

Serum samples of 113 patients with long bone fractures were collected over a period of 6 months following a standardised time schedule. TGF-β1 serum concentrations were measured using ELISA. Patients were assigned to 2 groups: Group 1 contained 103 patients with physiological healing. Group 2 contained 10 patients with impaired healing. Patients in both groups were matched. One patient of the group 2 had to be excluded because of missing match partner. In addition, fracture haematoma from 11 patients of group 1 was obtained to analyse local TGF-β1 concentrations. 33 volunteers donated serum which served as control.

Results

TGF-β1 serum concentrations increased during the early healing period and were significantly higher in patients with physiological healing compared to controls (P = 0.04). Thereafter, it decreased continuously between weeks 2 and 8 and fell again after week 8. TGF-β1 serum concentrations in patients with physiological healing were significantly higher at week 24 compared to controls (P = 0.05). In non-unions, serum concentrations differed significantly from those of controls at week 6 (P = 0.01). No significant difference in between patients with physiological and impaired fracture healing was observed. Fracture haematoma contained significantly higher TGF-β1 concentrations than peripheral serum of the patients (P = 0.017).

Conclusion

Elevated levels of TGF-β1 in haematoma and in serum after bone fracture especially during the entire healing process indicate its importance for fracture healing.     

Anti-GBM glomerulonephritis in mice lacking IL-1β-converting enzyme (ICE)

Background. Interleukin-1 (IL-1) has been reported to play a major role in the initiation and progression of several glomerulonephritis, and inhibition of IL-1 by the blockade of IL-1β-converting enzyme (ICE) has been suggested to be an ideal therapeutic strategy. Methods. To examine the effect of ICE inhibition on glomerulonephritis, we examined the susceptibility of ICE-deficient mice (ICE−/−) to anti-glomerular base-ment membrane antibody-induced glomerulonephritis (antiGBMGN), which has been previously reported to be mediated by IL-1β. Results. After the injection of antiGBM antibody to ICE−/− and wild type mice, albuminuria rose progressively and both groups of mice died within 7–9 days. Laboratory analysis of proteinuria, serum creatinine, and glomerular histology revealed no significant difference between the two groups. To pursue the mechanism of this result, bone marrow-derived monocyte/macrophage lineage cells (Mo/Mq cells) were established from both groups and the potency of IL-1β production in response to lipopolysaccharide was examined. An enzyme-linked immunosorbent assay (ELISA) of IL-1β revealed that, Mo/Mq cells from ICE−/− mice secreted IL-1β in response to lipopolysaccharide, although to a lesser extent than the Mo/Mq cells from ICE+/+ mice, suggesting that other protease(s) may process proIL-1β to generate the mature form. In fact, as was seen in the wild type mice, serum from antiGBM-injected ICE−/− mice contained IL-1β. Conclusions. These data suggest a limited effectiveness of ICE inhibition as a therapeutic strategy for glomerulonephritis. Received: May 18, 1999 / Accepted: October 25, 1999     

Laparoscopic approach of gastric gastrointestinal stromal tumors (GISTs): is it still a courageous choice? Report of two cases

Gastrointestinal stromal tumors (GISTs) are a well-defined clinicopathologic and molecular tumor entity, representing the most common gastrointestinal mesenchymal neoplasm. Differential diagnosis between GIST and other mesenchymal malignancies is crucial, given the successful management using targeted therapy in metastatic GIST. The mainstay of treatment remains surgery, complete tumor resection being the most important independent prognostic factor. Videolaparoscopic approach is still controversial for the high risk of tumor rupture or bleeding. Here we report 2 cases of GIST surgically resected using a videolaparoscopic approach and discuss the efficacy of this technique in selected patients.     

Epidemiology of testicular tumors in Slovakia (1993–1997): Preliminary report

Worldwide increase of the incidence of testicular tumors was also reflected in the increasing number of these malignancies in the Slovak Republic. Lack of the accurate information about the occurrence of testicular tumors in Slovakia has helped to create a new multicentric retrospective study based on occurrence, histology, risk factors, diagnosis and treatment of this malignancy in Slovakia. The analysed study group consists of 1010 patients with testicular cancer, diagnosed from the beginning of 1993 to the end of 1997. Identification and histological data about the patients were obtained from the heads of departments of urology in Slovakia. In this study considerable differences were found between information obtained from departments of urology and information published by the National Cancer Register of the Slovak Republic. Ascertained information is higher than the one published by the National Cancer Register from the last five officially concluded years.     

The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions.The protein tyrosine phosphatase (PTP)-like proteins islet antigen-2 (IA-2) and IA-2β are major type 1 diabetes autoantigens (1,2) localized to secretory granule membranes of islets and other neuroendocrine cells (3–5). Autoantibodies to the intracellular domains of IA-2 (IA-2ic) and IA-2β can be detected in the serum of 60–80% of patients with recent-onset type 1 diabetes (5–8) and in combination with antibodies to the other islet antigens—glutamate decarboxylase, zinc transporter 8, and insulin—they are valuable markers for predicting disease (9,10).IA-2 and IA-2β are transmembrane proteins of 979 and 1015 amino acids, respectively. The intracellular domains include a juxtamembrane (JM) region and a PTP-like region (7,11). Despite having no (IA-2) or only weak (IA-2β) phosphatase activity (2,12,13), their PTP-like regions share 88% amino acid sequence homology, including the core cysteine of the PTP signature motif (I/VHCXXGXXRS/T) essential for phosphatase activity in catalytically active members of the PTP protein family (14) (Fig. 1). In contrast, the JM regions of the two proteins share <50% homology. During the type 1 diabetes prodrome, autoantibodies to the PTP-like region may be preceded by a response to epitopes in the JM region but, as the disease progresses, reactivity spreads so that antibodies to PTP epitopes usually predominate (3,15). Autoantibodies bind to PTP epitopes common to both IA-2 and IA-2β; 50–80% of patient sera that react with IA-2 also recognize IA-2β, whereas 95% of sera that react with IA-2β also bind IA-2 (1,5,6,16,17).Open in a separate windowFIG. 1.Alignment of IA-2ic and IA-2β PTP sequences in pSP64 and pGEM, respectively. The IA-2 PTP sequence in pGEM is the same as IA-2ic, except it begins at amino acid 687 marked by the inverted triangle. Nonconserved amino acids are shaded. The highly conserved PTP core sequence is boxed (907–917) and differs by one amino acid between IA-2 and IA-2β. All 10 cysteines (boxed) in IA-2ic and cysteine at position at 945 in IA-2β were substituted with serine. Cysteine at position 909 and the corresponding cysteine in IA-2β (position 945) were changed to alanine. Glutamate at position 836 in IA-2 and 872 in IA-2β (circled) was changed to a lysine. Tryptophan at position 799 was changed to alanine in IA-2 PTP. The WPAE motif is also marked (boxed 875–878). The numbers above the sequence refer to the amino acid positions in IA-2ic. Sequences were obtained from GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"L18983","term_id":"662362","term_text":"L18983"}}L18983 (IA-2) and {"type":"entrez-nucleotide","attrs":{"text":"Y08569","term_id":"1644377","term_text":"Y08569"}}Y08569 (IA-2β).Two linear epitopes have been identified in the JM region [amino acids 611–620 and 621–630 (12,18)], but the conformational epitopes found in the PTP region are more diverse (6,19–22). PTP residues shown to be important for antibody binding include tryptophan 799, glutamate 836, asparagine 838, tyrosine 855, asparagine 858, and glutamine 862 (23–25). Studies of IA-2 crystal structure suggest that these residues form one epitope region (26). A second major PTP epitope region includes residues 876–880, which overlap the WPAE loop (25,27), as well as alanine 877 and aspartate 911, which are in close proximity (3).Disulfide-bond formation in the PTP region was thought to be critical for maintaining antigenic structure, as reduction and alkylation of cysteine residues abolished PTP autoantibody binding and removed resistance to digestion with trypsin (28). Analysis of IA-2ic expressed in Escherichia coli suggested the formation of at least one disulfide bridge (29), although its location was unknown. We also showed that azide and high concentrations of Tween-20 reduced IA-2A binding to the PTP domain (30), effects we ascribed to disruption of disulfide bonds and/or modification of the core cysteine. In other PTPs, this cysteine is susceptible to oxidation by reactive oxygen species (14,31). Oxidation inhibits PTP activity and is increasingly recognized as a mechanism for reversible regulation of PTP function (14,32), although the oxidized form of the core cysteine in IA-2 and IA-2β has yet to be determined.To investigate the influence of disulfide-bond formation on autoantibody binding and the importance of the core cysteines, C909 in IA-2 and C945 in IA-2β, we mutated individual cysteines within the cytoplasmic region of IA-2, IA-2 PTP, and IA-2β PTP and measured changes in antibody binding by radiobinding assay.