Piroxicam and Acarbose as Chemopreventive Agents for Spontaneous Intestinal Adenomas in APC Gene 1309 Knockout Mice

The use of nonsteroidal anti-inflammatory drugs has been suggested to have a chemopreventive effect against colon carcinoma, through the inhibition of cyclooxygenases 1 and 2, in patients with familial adenomatous polyposis and in animal models. Acarbose, an alpha-glycosidase inhibitor, may also be chemopreventive. In order to examine the effects of these drugs we employed APC gene knockout mice randomized into 3 groups, one for treatment with piroxicam (0.05% concentration in drinking water), one for acarbose (0.04% concentration in food) and another for the control. After 14 weeks of treatment, mice were killed for quantitation of gastric and intestinal adenomas. Tumor multiplicity in the whole gastrointestinal tract decreased from 33.89±13.07 tumors/mouse in the control group to 17.05±7 tumors/mouse in the piroxicam-treated group ( P <0.001). The decrease in the acarbose-treated group (29.68±12.86 tumors/mouse) was not significant ( P >0.05). The number of tumors ≥3 mm in diameter was also quantified in all gastrointestinal segments. The number of such tumors in the piroxicam group was decreased to 0.56±1.2 tumors/mouse from the control value of 3.78±1.17 tumors/mouse ( P <0.001), while in the acarbose-treated group the number decreased to 2.36±1.7 tumors/mouse ( P <0.01). Thus, piroxicam decreases the size and number of gastrointestinal adenomas in APC 1309 knockout mice, while acarbose decreases only the size.     

肝细胞癌中SFRP1和APC基因启动子甲基化与其mRNA的表达

目的探讨肝细胞癌（HCC）中SFRP1和APC基因启动子甲基化状态及其mRNA表达的关系。方法应用甲基化特异性聚合酶链反应（MSP）和逆转录-聚合酶链反应（RT—PCR）技术，检测30例肝细胞癌（HCC组）及相应的癌旁组织（癌旁组）和10例正常肝组织（正常对照组）中SFRP1及APC基因启动子甲基化状态和mRNA的表达水平，分析甲基化与某些临床参数与mRNA表达的关系。结果HCC组、癌旁组及正常对照组中的SFRP1和APC基因启动子甲基化率分别是11／30，4／30，0／10和14／30，5／30，0／10；HCC组明显高于其余两组（P〈0．05）。SFRP1基因启动子甲基化与临床资料无关（P〉0．05）；APC基因启动子甲基化与年龄（〈60）岁和癌肿无假包膜有关（P〈0．05）。HCC组SFRP1基因mRNA表达明显低于其余两组（P〈0．05），各组APC基因mRNA表达差异无显著性。两基因甲基化之间无相关性。结论SFRP1和APC基因启动子甲基化与HCC的形成和进展有关，但HCC组织中基因甲基化与mRNA表达的关系尚不明确。     

Novel APC-cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

Summary.  Background: Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined. Objective: To identify and characterize the additional APC-cleavage sites in FVa. Methods: The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV variants were expressed and their sensitivity for APC characterized functionally and with Western blotting. Results: The additional APC-cleavage sites were located at Lys309, Arg313, Arg316, Arg317 and Arg505. FVa-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q (denoted 8M-FVa) was APC resistant. To investigate individual sites, they were mutated back using 8M-FV as a template. The kinetics of APC-degradation of these variants demonstrated that protein S was equally efficient in enhancing the APC effect for all the novel sites. Conclusions: Multiple APC-cleavage sites close to Arg306 and a single site close to Arg506 were identified. Protein S was equally efficient as APC cofactor for all novel sites. The stimulation by protein S of the Arg505 cleavage argues against a specific protein S-mediated stimulation of cleavage at Arg306 due to relocation of the APC active site closer to the membrane.     

PLS—紫外分光光度法同时测定复方阿斯匹林片中三组分含量

本文研究了复方阿斯匹林片中阿斯匹林、非那西丁和咖啡因含量同时测定的紫外分光光度法的最佳实验条件,并简述偏最小二乘法(PLS)在多组分同时测定中的基本原理和应用。三组分模拟试样回收率平均值的置信区间分别为100.1±0.23%,100.0±0.25%和100.1±0.33%(置信度95%)。PLS法是一种理想的多组分测定方法,计算速度较快,结果更准确可靠,尤其适用于成批试样的分析,为微机控制的紫外可见分光光度计提供了一种新方法。     

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia

BACKGROUND: Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. OBJECTIVE: To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non-carriers with comparable prothrombin levels. PATIENTS/METHODS: Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. RESULTS: Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non-carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver-synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non-carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels. CONCLUSIONS: Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC-resistant than individuals with comparable prothrombin levels because of other causes.     

Immunoassay for wild-type protein in lymphocytes predicts germline mutations in patients at risk for hereditary colorectal cancer

Using colorectal cancer (CRC) as an example, we present the hypothesis that quantitative immunoassays for wild-type (full-length) proteins can be used to identify carriers of traits for hereditary diseases. In the case of hereditary CRC, this involves identifying individuals with germline mutations in a mismatch-repair (MMR) gene (mainly hMSH2 or hMLH1) or in the adenomatous polyposis coli (APC) gene. Because expression of wild-type protein should reflect wild-type gene dosage, we predicted that individuals harboring a germline mutation will have a reduction of approximately 50% in expression in lymphocytes of the corresponding full-length protein. In this pilot study, we tested lymphoblastoid cell lines that had been established from controls and individuals with, or at high risk for, hereditary CRC: 9 lines from healthy, unaffected individuals; 4 from affected members in familial adenomatous polyposis families (with known germ-line APC mutation); 42 from CRC patients in our Familial CRC Registry (increased risk of hereditary nonpolyposis colon cancer as assessed by family history, age at adenoma or carcinoma diagnosis, and other clinical criteria). For MSH2 and MLH1 we used western blots; for APC we used immunoprecipitation. All familial adenomatous polyposis lines had about 50% less immunoprecipitable full-length APC protein. Some cell lines (7 of 42) from Familial CRC Registry patients showed on western blots a reduction (mean 46%) in either MSH2 or MLH1 (relative to the other protein). All 7 subsequently were proved to contain a germline MMR mutation. We conclude that (1) because most of the expected CRC-causing germ line mutations are truncation-causing, immunoassays for wild-type protein should be able to identify most individuals with hereditary CRC-causing traits; (2) these assays, which are more practical and inexpensive than current mutation-detecting tests for hereditary CRC traits, have the potential for commercial development into broad-based population screens of high-risk patients and their families and the potential to save both lives and health-care dollars; (3) this strategy may be useful for other hereditary cancers and even other hereditary diseases; (4) our approach has the potential to greatly benefit public-health programs for cancer control.     

Human mast cells present antigen to autologous CD4+ T cells

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APC、P53基因突变与内镜下早期大肠癌分型的关系

目的　探讨内镜下早期大肠癌的分型与APC、P5 3基因突变的关系。方法　应用PCR SSCP方法对本院内镜下及病理诊断的5 4例早期大肠癌患者的APC及P5 3基因突变进行检测,并对内镜下的不同形态大肠早期癌分组进行比较。结果　隆起型(Ⅰ型)和表面隆起型(Ⅱa型)组与表面平坦型(Ⅱb型)、表面凹陷型(Ⅱc型)、还有混合型(Ⅱa Ⅱc和Ⅱc Ⅱa型)组进行比较,APC基因的突变率明显增高,存在显著差异(P <0 . 0 5 ) ,而P5 3基因的突变率在两组间没有显著差异。结论　我们推测平坦及凹陷型的表浅型早期大肠癌与隆起型有不同的基因突变途径。