Promoter 1A Research Articles

TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of non-Hodgkin lymphomas

AbstractThe constitutive activation of nuclear factor-κB (NF-κB) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-κB pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in Epstein-Barr virus–infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-κB activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-κB pathways may be advantageous for treatment.

Adenomatous polyposis coli 1A is likely to be methylated as a passenger in human gastric carcinogenesis

Many promoter CpG islands (CGIs) are methylated as a consequence of or in association with carcinogenesis (passenger), in addition to being a cause of carcinogenesis (driver). In gastric cancers, promoter 1A of the adenomatous polyposis coli ( APC) gene is frequently methylated, and is often discussed as a driver. However, the actual role of 1A methylation is unclear because the same APC protein is coded by two transcripts from two promoters, 1A and 1B, and their relative expression levels in gastric mucosae have not been quantified. To clarify this issue, we first identified detailed transcription start sites of 1A and 1B transcripts. We then confirmed that, among nine gastric cancer cell lines, 1A methylation, if present, could repress 1A transcription while 1B was expressed and not methylated. In primary samples, 1B expression was 15-fold higher than 1A expression in gastric mucosae of healthy volunteers, and was decreased markedly in non-cancerous gastric mucosae of cancer patients. Quantitative methylation analysis showed that promoter 1A was methylated at similar levels (20–40%) in healthy individuals and non-cancerous gastric mucosae of cancer patients, and promoter 1B was never methylated in any samples, including gastric cancers. These findings strongly indicated that methylation of APC promoter 1A is a passenger, and suggested that marked down-regulation of 1B expression could be related to formation of a field predisposed to gastric cancers.

Identification of cis-acting promoter sequences required for expression of the glycerol-3-phosphate acyltransferase 1 gene in mice

Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the “classical” sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/ db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.

Promoter hypermethylation leads to decreased APC mRNA expression in familial polyposis and sporadic colorectal tumours, but does not substitute for truncating mutations

Germline mutations in the tumour suppressor APC cause familial adenomatous polyposis (FAP), and somatic mutations are common in sporadic colorectal cancers (CRCs). Hypermethylation of APC promoter 1A has been reported in a substantial proportion of sporadic CRCs and may cause transcriptional silencing. Methylation has been proposed as an alternative to mutation or loss of heterozygosity as a mechanism of gene inactivation. However, the significance of APC methylation has remained unclear, because it has not previously been related to the presence of mono- or bi-allelic mutations at APC. We examined 103 FAP adenomas, 11 attenuated FAP adenomas, 31 sporadic CRCs and 30 CRC cell lines, all with known germline and/or somatic APC mutations. Overall, APC promoter 1A methylation was detected in 27–45% of colorectal tumours and cell lines, but generally not in histologically normal colorectum. In contrast to previous reports, methylation was detected in similar proportions of FAP/AFAP and sporadic CRCs. Importantly, methylation was independent of the presence, number and positions of APC mutations and was not associated with the CpG island methylator phenotype. Methylation resulted in a decrease or loss of 1A isoform mRNA and reduced total APC transcript levels, although expression was retained from promoter 1B. However, neither APC protein levels, nor transcription of a panel of Wnt target genes was associated with methylation status. Our data suggest that APC promoter 1A hypermethylation may influence APC expression levels in a subtle fashion, but methylation does not result in complete gene inactivation or act as a ‘second hit’.

Design of a zinc finger protein binding a sequence upstream of the A20 gene

BackgroundArtificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.ResultsIn order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.ConclusionIt is feasible to design specific zinc finger proteins by bioinformatics methods.

Interplay between E-box and NF-κB in Regulation of A20 Gene by DRB Sensitivity-inducing Factor (DSIF)

The NF-kappaB target gene A20 serves as a paradigm for gene-specific control of transcription elongation. This gene is regulated by the elongation factor DSIF (DRB sensitivity-inducing factor) under basal and NF-kappaB-activated states by two distinct mechanisms. Prior to NF-kappaB stimulation, the A20 gene is occupied by polymerase II, and elongation is inhibited by DSIF. This inhibition is mediated by an upstream promoter element termed ELIE (elongation inhibitory element). Upon NF-kappaB activation, inhibition of the A20 gene by DSIF persists, but now NF-kappaB and the core promoter regulate DSIF instead of ELIE. Here we investigated the regulation of DSIF by ELIE and the regulatory switch from ELIE to NF-kappaB following NF-kappaB induction. Electrophoretic mobility shift assays revealed two distinct protein complexes that specifically interact with ELIE, one of which is the E-box protein USF1. Interestingly, USF1 is displaced from the A20 promoter upon induction of NF-kappaB. A mutation in the E-box section of ELIE diminished the binding of USF1 and DSIF recruitment. Consistent with these findings, the E-box is crucial for DSIF inhibition in resting, but not NF-kappaB-stimulated, cells. These findings reveal a dynamic regulation of DSIF involving either E-box or NF-kappaB depending on the physiological circumstances.

Role of Nucleosomal Occupancy in the Epigenetic Silencing of the MLH1 CpG Island

Epigenetic silencing of tumor suppressor genes is generally thought to involve DNA cytosine methylation, covalent modifications of histones, and chromatin compaction. Here, we show that silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells involves distinct changes in nucleosomal occupancy. Three nucleosomes, almost completely absent from the start sites in normal cells, are present on the methylated and silenced promoter, suggesting that epigenetic silencing may be accomplished by the stable placement of nucleosomes into previously vacant positions. Activation of the promoter by demethylation with 5-aza-2'-deoxycytidine involves nucleosome eviction. Epigenetic silencing of tumor suppressor genes may involve heritable changes in nucleosome occupancy enabled by cytosine methylation.

Profiling YB-1 responsive genes in basal-like breast cancer cells by ChIP-on-chip reveals direct binding to PIK3CAs

Purpose: Basal-like breast cancers (BLBC) are typically very aggressive and they are also prone to a high rate of recurrence. We recently identified the transcription factor Y-box binding protein-1 (YB-1) as an important component of BLBC by screening breast tumour tissue microarrays. YB-1 is well known for its ability to confer chemotherapy resistance and to promote the growth of a wide range of cancer cell types. The breast cancer cell line SUM149 was characterized by us as having all the hallmarks of BLBC including a lack of the estrogen, progesterone, and Her-2 receptors. We also find that they express high levels of the epidermal growth factor receptor, a YB-1 responsive gene. 
 We performed a genome-wide promoter screen of the possible YB-1 responsive genes using the NimbleGen ChIP-on-chip (COC) platform to begin to understand how this oncogene regulates the growth of basal-like breast cancer cells. YB-1 was found to bind to ~6,700 human promoters. Several members of the phosphatidylinositol-3-kinase (PI3K) pathway were identified (p110a, p85, Akt-2, ILK). Of particular interest was the identification of PIK3CA, the gene that codes for the p110a catalytic subunit of PI3K. PI3K, a lipid kinase with important roles in neoplasia, phosphorylates membrane inositol lipids resulting in activation of downstream effectors that are involved in cellular responses such as cell proliferation, survival, metabolism, and cytoskeletal reorganization. Many studies have suggested importance of the role of PI3K signaling in various types of cancer. Approximately 20% of breast cancers harbour somatic activating mutations in PIK3CA, and about 8% have amplification of PIK3CA. The remainder of the cancers have elevated levels of PI3K via other mechanisms including increased transcription. 
 Methods & Results: Here we propose that YB-1 overexpression in cancer leads to increased PIK3CA transcription via direct binding to the promoter. To extend our COC findings, we knocked down YB-1 with siRNA and observed a concomitant loss in p110a by immunoblotting. We have recently identified two alternate PIK3CA transcriptional start sites (exon 1a and 1b) and two alternate PIK3CA promoters (promoter 1a and 1b). We mapped potential YB-1 binding sites on PIK3CA promoters 1a and 1b, and identified five putative YB-1 binding sites within promoter 1a and one putative binding site within promoter 1b. We further performed traditional ChIP and EMSA to confirm the interaction of YB-1 with the putative binding sites on PIK3CA promoter, and determined that YB-1 binds to promoter 1a, but not to promoter 1b. 
 Conclusion:The demonstration that YB-1 binds directly to the PIK3CA promoter and induces its activity identifies a novel mechanism whereby activation of YB-1 and subsequent up-regulation of PIK3CA may contribute to the pathophysiology, outcomes and therapeutic responsiveness of breast cancer.

Promoter methylation and mRNA expression of APC gene in MCF10 breast cancer model

To investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression. Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ), MCF10CA1a, MCF10CA1d, MCF10CA1h cell lines (invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential). In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays. Hypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS.com, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS.com showed reduced mRNA expressions of APC at 0.27, 0.96, 1.78, 2.70, and 2.03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0.02 and 0.33 times, respectively). The aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.

Large BRCA1 deletion in African Americans

Large genomic rearrangements have been observed in BRCA1 carriers and frequently involve recombination between intron Alu sequences, leading to a non-functional protein. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can detect the change in copy number of the 24 exons of BRCA1 simultaneously in one reaction. A large deletion of exons 1a, 1b, and 2 has been detected in triplicate MLPA analyses of one (family #70) of our 55 high-risk African American breast cancer families. The BRCA1 Pro871Leu polymorphism is usually in linkage disequilibrium with the BRCA1 β promoter C/G 1802 polymorphism. Linkage of the Pro871Leu polymorphism to the β promoter C/G 1802 polymorphism was tested using specific β promoter primers. In family #70, the Pro871Leu polymorphism was linked to a hemizygous/homozygous C in the β promoter. The hemizygosity of the BRCA1 β promoter 1802 polymorphism suggests that the deletion of exons 1a, 1b, and 2 extends into the promoter region. A 37 kb deletion has been reported in Caucasians that removes the BRCA1 promoter and BRCA1 introns 1a, 1b, and 2 resulting in no BRCA1 transcript. Due to the 98% identity of sequences in BRCA1 intron 2 and Ψ-BRCA1 intron2, this region is a hot spot for recombination. This is the first report of a large deletion of exons 1a, 1b, and 2 in an African American family. Supported by U.S. Army grant DAMD17-01-1-0266.

Nuclear Factor-κB Regulates β-Cell Death

Apoptotic β-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of β-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized β-cells to tumor necrosis factor (TNF)-α–induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in β-cells. Regulation of A20 was nuclear factor-κB (NF-κB)–dependent, two NF-κB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in β-cells. Activation of NF-κB by TNF receptor–associated factor (TRAF) 2, TRAF6, NF-κB–inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect β-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in β-cells. Further, A20 expression is NF-κB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis.

Study of the methylated CpG status of adenomatous polyposis coli gene promoter 1A sequence on lung cancer

目的 探寻结肠多发性腺瘤样息肉(APC)基因启动子1A序列中肺癌甲基化位点及其存在模式.方法用甲基化序列特异性引物,对阳性控制样品(CB+)、阴性控制样品(CB-)、13份正常肺组织及19份肺癌组织进行APC基因启动子1A序列甲基化特异性扩增(MSP),其扩增产物直接测序和进行克隆测序分析.结果在19份肺癌组织中,APC基因启动子1A序列的707、714、719和726位点发生高甲基化,甲基化率分别为95%(18/19)、74%(14/19)、74%(14/19)和74%(14/19),与正常肺组织比较,差异均有统计学意义(P＜0.001).而679和687位点甲基化发生率均为11%(2/19),与正常肺组织甲基化发生率(0/13)接近(P＞0.05).结论在APC基因启动子1A序列中存在CpG胞嘧啶二核苷酸特定甲基化模式,其对肺癌早期诊断可能有重要意义。

Cytoplasmic expression of E‐cadherin and β‐Catenin correlated with LOH and hypermethylation of the APC gene in oral squamous cell carcinomas

Inactivation of the adenomatous polyposis coli (APC) gene results in accumulation and translocation of beta-Catenin, which are important for malignant development. The aim of the present study is to investigate the possible role of APC/beta-Catenin pathway in oral squamous cell carcinomas. The DNA from 34 patients was examined for loss of heterozygosity (LOH) at two markers surrounding the APC, and for hypermethylation of the APC promoter by using methylation-specific polymerase chain reaction (MS-PCR). Fifteen of 34 samples were stained immunohistochemically to show the expression of E-cadherin and beta-Catenin. We found that cytoplasmic rather than membrane staining of E-cadherin and beta-Catenin was a prominent aberrant tumour-related alteration, and that this expression was mainly present in moderately and poorly differentiated tumours. LOH and hypermethylation of the APC promoter was found in four of 31 and five of 34 carcinoma samples, respectively. Four of five cases presenting LOH/hypermethylation showed cytoplasmic expression of E-cadherin and beta-Catenin by immunohistochemical (IHC) staining. The present results indicate that LOH at the APC locus or hypermethylation of the APC promoter 1a may lead to free beta-Catenin accumulation in cytoplasm of oral carcinoma cells and thereby to oral malignant progression.

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.

Regulation of SCL Expression by the Homeodomain Protein Otx-1 and the Erythroid Transcription Factor GATA-1.

Members of the bicoid homeodomain-containing proteins are important in establishing left-right asymmetry and the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that Otx1, a member of the bicoid homeodomain-containing proteins, is co-expressed with the SCL transcription factor in hematopoietic pluripotent and erythroid progenitor cells and Otx1-deficiency impairs the erythroid compartment in mice, associated with decreased SCL levels. In the present study, we provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. First, we show by chromatin immunoprecipitation that Otx1 and GATA-1 are specifically bound to the SCL proximal promoter in erythroid cells. Second, Otx-1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity depends on the integrity of the proximal GATA site of the SCL promoter 1a. At the molecular level, we show that this synergy occurs via a physical interaction between Otx-1 and GATA-1 in erythroid cells, which maps to the homeodomain of Otx-1. Furthermore, a gain of function of Otx1 in primary hematopoietic cells gives rise to a 6-fold increase in endogenous SCL levels, an increase in TER119-positive erythroid cells and a decrease in the number of CD11b-positive myeloid cells. Finally, a gain of function of SCL rescues the erythroid deficiency in Otx1−/− mice, consistent with the view that SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL operate within the same genetic pathway to specify the erythroid fate during hematopoiesis.

Detection of Tumor Markers Including Carcinoembryonic Antigen, APC, and Cyclin D2 in Fine-Needle Aspiration Fluid of Breast

The traditional triple test for breast cancer diagnosis is physical examination, mammography, and aspiration cytology. However, the accuracy of mammography on young women with nonatrophied breasts is poor compared with that for women older than 50 years, and additional methods for diagnosis of breast cancer are needed. To investigate whether carcinoembryonic antigen (CEA), CA 15-3, and CA 125 concentrations in breast aspiration fluid are useful as breast cancer biochemical markers and whether APC and cyclin D2 gene promoter hypermethylation could be regarded as a breast cancer molecular marker. CEA, CA 15-3, and CA 125 concentrations were measured, and methylation status of the APC gene promoter 1A and the promoter region of the cyclin D2 gene were analyzed using a methylation-specific polymerase chain reaction assay of ex vivo breast aspiration fluid obtained from 49 samples of excised breast tissue. The specimens were collected during a 1-year period in the tertiary care teaching hospital in Seoul, Korea. Forty-nine patients with breast masses were surgically treated. Thirty-four patients had breast cancer, and 15 had benign breast disease. Aspiration fluid CEA concentrations were significantly higher in breast cancer cases than in cases of benign breast disease (mean, 69.90 ng/mg protein vs 0.68 ng/mg protein, respectively; P < .001). At 90% specificity of the assay (CEA, 2.13 ng/mg protein), the corresponding sensitivity for breast cancer detection was 62%, according to the receiver operating characteristic curve drawn. The APC gene promoter 1A and the promoter region of the cyclin D2 gene were methylated in 42% (14/33) and 70% (23/33) of the breast cancer aspiration fluid samples, respectively. A cumulative incidence of methylation of these 2 genes was 85% (28/33). The APC and cyclin D2 gene promoters were both unmethylated in the aspiration fluids from 19 women with nonmalignant breast disease. Breast aspiration fluid CEA concentration and the methylation of the APC gene promoter 1A and the promoter region of the cyclin D2 gene can be used as tumor markers to overcome some of the limitations of aspiration cytology. In combination with the mammogram and physical examination, assays for these markers could be used to help determine a definitive diagnosis when cytologic results are suspicious for malignancy.

Three mechanisms are involved in glucocorticoid receptor autoregulation in a human T-lymphoblast cell line.

Glucocorticoids up-regulate the glucocorticoid receptor (GR) in the human T-lymphoblast cell line CEM-C7. One mechanism for the up-regulation of the GR protein is the well-known up-regulation of GR transcripts. We have investigated the effect of other factors on the up-regulation. At least three promoters (1A, 1B, and 1C) exist, which give rise to GR transcripts with different exon 1 sequences. Transcripts with different exon 1 sequences have similar stabilities. Glucocorticoids have little, if any, effect on mRNA stability. In transfection experiments of the GR-deficient mouse fibroblast cell line E8.2, different exon 1 sequences furthermore caused no significant differences in the translational efficiencies of GR transcripts. However, the ratio between the concentrations of the glucocorticoid receptor B (GR-B) isoform and the glucocorticoid receptor A (GR-A) isoform was higher for transcripts containing the exon 1A3 sequence arising from promoter 1A than in transcripts containing exon 1 sequences from promoters 1B and 1C. Because the GR-B isoform is more active in transactivation then GR-A, this would tend to fine-tune glucocorticoid responsiveness of CEM-C7 cells, which express exon 1A3-containing transcripts. We also found that glucocorticoids do not decrease the stability of the GR protein in CEM-C7 cells. In contrast to other cell lines that downregulate GR expression in response to glucocorticoids, CEM-C7 lymphoblasts possess three mechanisms ensuring high glucocorticoid responsiveness: an up-regulation of GR mRNA by glucocorticoids, no destabilization of GR protein by glucocorticoids, and a high activity of promoter 1A with concomitant high expression of the GR-B isoform.

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Genetic and epigenetic alterations of the APC gene in malignant melanoma.

High levels of beta-catenin and activating mutations in the beta-catenin gene (CTNNB1) have been demonstrated in malignant melanomas, implicating dysregulated Wnt signalling in the pathogenesis of this malignancy. We systematically examined melanoma cell lines for activating CTNNB1 mutations as well as genetic and epigenetic alterations of the adenomatous polyposis coli gene (APC), another key component of the Wnt signalling transduction pathway. Of 40 cell lines tested, one carried a truncating APC mutation and loss of the corresponding wild-type allele, and one carried a CTNNB1 missense mutation. Hypermethylation of APC promoter 1A was present in five of the cell lines (13%) and in nine of 54 melanoma biopsies (17%). Cells with truncating APC or activating CTNNB1 mutations showed increased transcription from endogenous and ectopic beta-catenin/T-cell factor (Tcf)-responsive target genes, consistent with the known effects of these alterations on beta-catenin stability and Tcf transactivation. In contrast, cell lines with APC promoter 1A hypermethylation did not show increased Wnt signalling, probably due to residual APC activity expressed from promoter 1B. Suppression of APC transcripts in melanoma cells by stable expression of short hairpin RNAs led to a Wnt signalling-independent increase in cell proliferation, but also reduced the invasive growth in collagen type I. Collectively, our data suggest that the tumour-suppressive function of APC in melanocytic cells is dose dependent. We propose that epigenetic silencing of promoter 1A may contribute to the development of malignant melanoma by reducing the expression of APC to a level that promotes cell proliferation without compromising the invasive capacity.

Aberrant methylation of the adenomatous polyposis coli promoter 1A in bronchial aspirates from patients with suspected lung cancer.

Promoter hypermethylation is a major mechanism for gene silencing and offers a promising starting point for developing molecular biomarkers. The purpose of our study was to determine aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A with respect to its prevalence and quantitative level in bronchial aspirates from patients with suspected lung cancer. Applying quantitative methylation-specific PCR, 155 bronchial aspirates from patients with non-small cell cancer (NSCLC) and small cell cancer (SCLC) of the lung as well as 67 bronchial aspirates from patients diagnosed for nonneoplastic lung disease were examined in a retrospective case-control study. Aberrant APC promoter 1A methylation was seen in 71% of NSCLCs, 38% of SCLCs and 42% of patients with nonneoplastic lung disease, being therefore not specific for the presence of primary lung cancer. In contrast, quantitative analysis showed a significantly higher methylation level of bronchial aspirates from NSCLC as compared to patients without neoplastic lung disease. Introducing a cutoff point that defined high level of APC hypermethylation NSCLC could be discriminated from cases without neoplastic disease with a specificity of 98.5% and a sensitivity of 39%. The data suggest that quantitative analysis of APC hypermethylation may serve as a biomarker of primary lung cancer.