乳腺癌APC基因甲基化状况及其蛋白表达

背景与目的:结肠腺瘤性息肉病基因(adenomatous polyposis coli,APC)蛋白表达产物是Wnt信号转导途径重要组成部分,该基因失活使β-catenin蛋白降解障碍,从而使Tcf/Lef激活,引起基因异常转录,最终产生癌变.启动子区甲基化是导致抑癌基因转录失活的重要机制.本研究探讨乳腺癌APC基因启动子1A区甲基化状态与其蛋白表达的关系,并分析APC基因异常甲基化与乳腺癌临床病理特征的关系.方法:应用甲基化特异性PCR和免疫组织化学方法检测76例乳腺癌及相应癌旁乳腺组织中APC基因启动子1A区甲基化状态以及蛋白表达.结果:癌旁乳腺组织中均未发现APC基因启动子1A区甲基化,乳腺癌组织中APC基因启动子1A区甲基化率为36.8%.癌旁乳腺组织中APC蛋白阳性率为100%,乳腺癌中APC蛋白阳性率为52.4%.乳腺癌组织中APG基因甲基化与APC蛋白表达呈负相关(r=-0.351,P＜0.05),与TNM分期呈正相关(r=0.335,P＜0.05).结论:乳腺癌发展过程中APC基因启动子1A区出现异常甲基化,是导致该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一.     

Recruitment of adenomatous polyposis coli and beta-catenin to axin-puncta

The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC- and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits beta-catenin destruction complex proteins, including APC, beta-catenin, glycogen synthase kinase-3-beta (GSK3-beta) and casein kinase-1-alpha (CK1-alpha). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for beta-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for beta-catenin degradation. An axinDeltaDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of beta-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for beta-catenin destruction.     

Beta-catenin and adenomatous polyposis coli (APC) mutations in adenomas from hereditary non-polyposis colorectal cancer patients

It is known that cisplatin (CDDP) potentiates the cytotoxicity of 5-fluorouracil (5-FU), and that the biochemical mechanism is an increase in the intracellular reduced folate levels in the tumor cells. We investigated the effect of consecutive administration with lower-dose CDDP on intracellular accumulation of reduced folate and the activity of methionine synthase, a key enzyme in intracellular methionine synthesis. When CDDP (1 mg/kg) was administered i.p. to ascitic Yoshida sarcoma-bearing rats for 4 consecutive days, both the reduced folate levels and methionine synthase activity in the cells significantly increased, as the same as a single 5 mg/kg dose of CDDP. Furthermore, when Yoshida sarcoma-bearing rats were pre-treated with 1 mg/kg CDDP for 5 consecutive days, [14C]L-methionine incorporation into the isolated ascitic cells was significantly inhibited as compared to that in non-treated cells, suggesting that consecutive administration of lower-dose CDDP is capable of inducing the intracellular modulation of reduced folate levels and methionine synthase activity via inhibition of cellular uptake of methionine. In addition, 5-day administration of lower-dose (1 mg/kg) CDDP potentiated the antitumor effect of 5 mg/kg S-1, a new oral preparation of tegafur, given for 7 consecutive days, and this combined effect was almost similar to the antitumor effect of a combination of S-1 and a single conventional dose (5 mg/kg) of CDDP. Consecutive lower-dose CDDP also may be concluded to act as an important modulator of the enhancement of 5-FU cytotoxicity in experimental tumors.     

Methylation of the adenomatous polyposis coli (APC) gene in human placenta and hypermethylation in choriocarcinoma cells

Methylation of the human APC gene promoter is associated with several different types of cancers and has also been documented in some pre-cancerous tissues. We have examined the methylation of APC gene promoters in human placenta and choriocarcinoma cells. This revealed a general hypomethylation of the APC-1b promoter and a pattern with monoallelic methylation of the APC-1a promoter in full term placental tissue. However, there was no evidence of a parent-of-origin effect, suggesting random post zygotic origin of methylation. Increased methylation of this promoter was observed in all choriocarcinoma-derived trophoblast cell lines, suggesting a trophoblastic origin of placental APC methylation and implicating APC hypermethylation in the development of this group of gestational tumours. Our demonstration of placental methylation of the APC-1a promoter represents the first observation of monoallelic methylation of this gene in early development, and provides further support for a role of canonical Wnt signalling in placental trophoblast invasiveness. This also implicates tumour suppressor gene silencing as an integral part of normal human placental development.     

肺癌细胞株APC基因启动子甲基化对其转录的影响

背景与目的:抑癌基因家族性腺瘤样结肠息肉病易感基因(adenomatous polyposis coli,APC)启动子区的高甲基化在很多肿瘤中被发现,与这些肿瘤的发生发展相关.本实验室在肺癌患者肿瘤组织中检测到APC甲基化率达47%,为了研究其在肺癌细胞株中的甲基化情况,并进一步了解甲基化对其转录的影响,本研究检测了3株肺癌细胞的抑癌基因APC启动子甲基化状态及其对该基因转录水平的影响.方法:提取3株肺癌细胞株(肺腺癌细胞株SPC-A1、小细胞肺癌细胞株NCI-H446、大细胞肺癌细胞株NCI-H460)的DNA,以经转甲基处理和未做处理的脐带血DNA为阳性、阴性对照,亚硫酸氢盐化学修饰后,用甲基化特异性基因扩增(methylation specific-polymerase chain reaction,MSP)和甲基化基因芯片对APC基因启动子1A CpG岛甲基化进行研究,并用实时荧光定量PCR(real time-polymerase chain reaction)技术,以Sybr-GreenⅠ为荧光染料,β-actin基因为内参照,检测mRNA转录;对甲基化阳性的NCI-H460细胞,分别用1、5、10、15 μmol/L的5'-杂氮-2'-脱氧胞嘧啶(5-aza-2-deoxycytidine,5-aza-dC)试剂进行脱甲基化,提取RNA,荧光定量检测其转录变化.结果:SPC-A1和NCI-H446细胞APC甲基化阴性,NCI-H460细胞APC甲基化阳性;甲基化芯片检测NCI-H460细胞在APC启动子1A 5个CpG位点均存在甲基化(687、707、714、719、726),SPC-A1和NCI-H446甲基化阴性,荧光定量结果NCI-H460的APC转录较SPC-A1和NCI-H446有明显的下降,仅为二者平均的30.04%;经5-aza-dC脱甲基化作用后,NCI-H460细胞的APC表达增加了约5～10倍,其中10 μmol/L浓度作用下,APC表达增加最多.结论:肺癌细胞株NCI-H460中存在APC基因高甲基化,5-aza-dC脱甲基化试剂可以激活其转录.     

The role of the adenomatous polyposis coli (APC) in oral squamous cell carcinoma

The main cause of death in oral squamous cell carcinomas (OSCC) is metastasis. Intercellular adhesion is mediated by a family of glycoproteins called cadherins and other molecules like catenins and APC (adenomatous polyposis coli) among other. The WNT (wingless-type) gene family is a group of genes, key signaling pathway for embryonic development and oncogenesis. The goal of this paper is to describe the role of the APC gene, and its derivatives, in the carcinogenicity pathway of WNT-1, identifying its role as a tumor suppressor gene in OSCC, while describing the genetic (loss of heterozygosity and mutations) and epigenetic alterations that modulate its expression and evaluate its relationship with the clinicopathological parameters of this type of tumors. As for APC, its activity as a tumor suppressor gene appears muted on a relatively frequent basis in these tumors, either by LOH, mutations or epigenetic control mechanisms, thus resulting in a low degree of agreement between the results of different studies.     

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.     

Impact of adenomatous polyposis coli (APC) tumor supressor gene in human colon cancer cell lines on cell cycle arrest by apigenin

This research assessed the importance of the adenomatous polyposis coli (APC) tumor suppressor mutation in the ability of apigenin to induce cell cycle arrest using HT29-APC cells, which contain inducible wild-type APC under the metallothionein promoter. HT29-GAL cells, containing beta-galactosidase (GAL), were included as control. Treatment with apigenin (0, 20, 40, 60, and 80 microM) for 48 h resulted in reduction in the cell number (P < 0.05) concurrent with flow cytometry results showing a dose-dependent accumulation of cells in the G2/M phase in both HT29-APC and HT29-GAL cells without ZnCl(2) treatment. Flow cytometric analysis showed an increase (P < 0.05) in the percentage of cells in G2/M when HT29-APC cells were treated with 80 microM apigenin for 120 h. This increase was not present in HT29-APC cells when treated with both 80 microM apigenin and 100 microM ZnCl(2) for 120 h. Western blot analysis verified the induction of APC protein expression in ZnCl(2)-treated HT29-APC cells but not in ZnCl(2)-treated HT29-GAL cells. Apigenin plus ZnCl(2) treatment increased the expression of APC protein in HT29-APC cells by 50 fold above expression observed with ZnCl(2) alone. Upon induction of the APC gene with ZnCl(2) in HT29-APC cells, the percentage of apoptotic cells increased significantly (P < 0.05) after 120-h treatment. Additionally, apigenin treatment (80 microM) further increased the percentage of apopototic HT29-APC following ZnCl(2) treatment to induce wild-type APC expression. These results suggest that APC dysfunction may be critical for apigenin to induce cell cycle arrest in human colon cancer cell lines and furthermore, apigenin enhances APC expression and apoptosis in cells with wild-type APC.     

Polymorphisms in the adenomatous polyposis coli (APC) gene and advanced colorectal adenoma risk

While germline mutations in the adenomatous polyposis coli (APC) gene cause the hereditary colon cancer syndrome (familial adenomatous polyposis (FAP)), the role of common germline APC variants in sporadic adenomatous polyposis remains unclear. We studied the association of eight APC single nucleotide polymorphisms (SNPs), possibly associated with functional consequences, and previously identified gene–environment (dietary fat intake and hormone replacement therapy (HRT) use) interactions, in relation to advanced colorectal adenoma in 758 cases and 767 sex- and race-matched controls, randomly selected from the screening arm of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Cases had at least one verified advanced adenoma of the distal colon; controls, a negative sigmoidoscopy. We did not observe an association between genotypes for any of the eight APC SNPs and advanced distal adenoma risk (P_{global gene-based} = 0.92). Frequencies of identified common haplotypes did not differ between cases and controls (P_{global haplotype test} = 0.97). However, the risk for advanced distal adenoma was threefold higher for one rare haplotype (cases: 2.7%; controls: 1.6%) (odds ratio (OR) = 3.27; 95% confidence interval (CI) = 1.08–9.88). The genetic association between D1822V and advanced distal adenoma was confined to persons consuming a high-fat diet (P_interaction = 0.03). Similar interactions were not observed with HRT use. In our large, nested case-control study of advanced distal adenoma and clinically verified adenoma-free controls, we observed no association between specific APC SNPs and advanced adenoma. Fat intake modified the APC D1822V-adenoma association, but further studies are warranted.     

Mutations of adenomatous polyposis coli (APC) gene are uncommon in sporadic desmoid tumours.

Desmoids are locally aggressive, non-metastasizing soft-tissue tumours, whose aetiology is still unclear. In patients affected with familial adenomatous polyposis (FAP), the incidence of desmoids is much higher than in the general population. The APC gene, which is responsible for FAP, is involved in the development of desmoids associated with this syndrome. In this study 16 sporadic and four FAP-related desmoids were analysed in order to investigate the possible involvement of APC in non-syndromic cases also. The 5'' end (exons 1-11) and the coding portion of exon 15 of APC were screened using the in vitro synthesized-protein assay (IVSP). Exons 5, 6, 8-14, and a region of exon 15 spanning codons 1036-1634 were investigated by single-strand conformation polymorphism (SSCP) analysis. APC germline mutations were identified in all FAP patients, but not in sporadic cases. Somatic mutations were found in three FAP-associated desmoids (75%) and two sporadic tumours (12.5%). In one of the latter cases, both alleles were affected. These findings indicate a limited role of the gene in the development of desmoid tumours outside FAP.     

The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression

Inactivation of the adenomatous polyposis coli (APC) gene is a critical event in the development of human colorectal cancers. At the biochemical level, several functions have been assigned to the multidomain APC protein, but the cellular effects of APC expression and how they relate to its biochemical functions are less well defined. To address these issues, we generated a recombinant adenovirus (Ad-CBR) that constitutively expresses the central third of APC, which includes all of the known beta-catenin binding repeats. When expressed in colon cancer cells, Ad-CBR blocked the nuclear translocation of beta-catenin and inhibited beta-catenin/Tcf-4-mediated transactivation. Accordingly, expression of endogenous targets of the APC/beta-catenin/Tcf-4 pathway was down-regulated. Ad-CBR infection of colorectal cancer cell lines with mutant APC but wild-type beta-catenin resulted in substantial growth arrest followed by apoptosis. These effects were attenuated in lines with wild-type APC but with mutated beta-catenin. These findings suggest that the beta-catenin-binding domain in the central third of APC is sufficient for its tumor suppressor activity.     

APC truncation and increased beta-catenin levels in a human breast cancer cell line

Mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene or the beta-catenin gene are present in most colon cancers and less frequently in other tumor types. In this study, we screened 24 human breast cancer cell lines and three immortalized human breast epithelial cell lines for alterations in beta- and gamma-catenin and APC by western blotting, protein truncation assay and DNA sequence analysis. In one cell line (DU 4475), an APC mutation was identified (E1577stop) that resulted in expression of truncated APC. This mutation was associated with elevated cytosolic beta-catenin levels, probably due to loss of APC function, as in colon cancers. No mutations were found in exon 3 of the beta- or gamma-catenin genes. We conclude that APC mutations and beta-catenin upregulation may occur with low frequency in human breast cancer cells.     

Immunochemical identification of novel high-molecular-weight protein isoforms of the adenomatous polyposis coli (APC) gene

Mapping analyses of monoclonal antibodies (MAbs) directed against the amino-terminus of the adenomatous polyposis coli (APC) gene product revealed that epitopes recognized by the MAbs FE9, CF11 and AC4 constitute different peptide sequences encoded by the APC exons 1, 2 and 3, respectively. The protein pattern detected with these specificity-defined immunoreagents, however, differed depending on the particular antibody used on Western blots of cellular urea extracts. APC exon 15-positive “classic” p300^apc polypeptide chains were identified by the MAb FE9, MAb CF11 and the C-terminus-specific MAb IEI, but only weak signals were obtained with the AC4 MAb, which defines an exon 3-encoded epitope. In contrast with this immunoreactivity, 2 novel high m.w. products of approx. 150/160 and 200 kDa were exclusively recognized by the AC4 MAb, which was shown to bind to the APC exon 3-encoded peptide sequence SRESTGYL. A molecular form of some 400 kDa was identified to represent a disulfide-bound oligomer of the p150/160^apc molecules. The novel APC-related molecules did not contain exon 1- and exon 15-encoded epitopes, as confirmed with the help of the FE9 and IE1 MAbs, respectively. This observation was corroborated by the fact that these novel proteins were not truncated in a collection of familial adenomatous polyposis patients with stop mutations in exon 15. We conclude, that APC MAb AC4-reactive p150/160 and p200 polypeptide chains represent novel genuine products of the APC gene devoid of exon 1- and exon 15-encoded protein interaction domains. © 1996 Wiley-Liss, Inc.     

Methylation analysis of the adenomatous polyposis coli (APC) gene in adult T-cell leukemia/lymphoma

We investigated methylation status of the adenomatous polyposis coli (APC) gene in adult T-cell leukemia/lymphoma (ATL). APC methylation was found in 15 of 31 (48%) primary samples, and 2 of 4 (50%) ATL cell lines. Methylation of the APC gene occurred more frequently in acute ATL (12/21) (57%) than chronic ATL (1/8) (13%) (P = 0.03). APC was not expressed in the APC-methylated ATL cell line ST1. Demethylation with 5-azacytidine treatment restored APC expression in the ST1 cell line. Our data show that hypermethylation of the APC gene is involved in the pathogenesis of ATL.     

Aberrant beta-catenin expression and adenomatous polyposis coli gene mutation in ameloblastoma and odontogenic carcinoma

The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC (adenomatous polyposis coli), beta-catenin and Axin are frequently mutated in some primary human cancers. This study was conducted to clarify the relation of beta-catenin accumulation and the mutation of the CTNNB1 (beta-catenin) gene with the mutation of APC gene in the process of development of odontogenic tumors including ameloblastoma and odontogenic carcinoma (OC). beta-Catenin accumulation was examined by immunohistochemistry in formalin-fixed, paraffin-embedded samples of six ameloblastomas and eight OCs. We also performed a mutation analysis of CTNNB1 and APC to examine the cause of beta-catenin accumulation. All ameloblastoma cases and six out of eight (75%) OC cases exhibited beta-catenin accumulation in the nucleus. CTNNB1 mutation was only found in one OC case, whereas three of six (50%) ameloblastoma cases and two out of eight (25%) OC cases had APC mutations within the mutational cluster region. Our findings suggest that aberrant beta-catenin expression and APC missense mutation may play an important role for the pathogenesis of epithelial odontogenic tumors.