cytidylyl-3--5--guanosine and Breast-Neoplasms

Hormone therapy is the most effective treatment for patients with estrogen receptor α-positive breast cancers. However, although resistance occurs during treatment in some cases and often reflects changed estrogen receptor α status, the relationship between changes in estrogen receptor α expression and resistance to therapy are poorly understood. In this study, we identified a mechanism for altered estrogen receptor α expression during disease progression and acquired hormone therapy resistance in aromatase inhibitor-resistant breast cancer cell lines. Subsequently, we investigated promoter switching and DNA methylation status of the estrogen receptor α promoter, and found marked changes of methylation at a single CpG site (CpG4) in resistant cells. In addition, luciferase reporter assays showed reduced transcriptional activity from this methylated CpG site. This CpG region was also completely conserved among species, suggesting that it acts as a methylation-sensitive Ets-2 transcription factor binding site, as confirmed using chromatin immunoprecipitation assays. In estrogen receptor α-positive tumors, CpG4 methylation levels were inversely correlated with estrogen receptor α expression status, suggesting that single CpG site plays an important role in the regulation of estrogen receptor α transcription.

Topics: Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Base Sequence; Breast Neoplasms; Conserved Sequence; Dinucleoside Phosphates; DNA Methylation; DNA, Recombinant; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Genes, Reporter; Humans; MCF-7 Cells; Middle Aged; Mutation; Neoplasm Proteins; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-2; Recombinant Proteins; Response Elements; Transcription, Genetic

The epidermal growth factor receptor (EGFR) is a member of the HER/ERB-B family of transmembrane receptor kinases. Overexpression of EGFR confers advantages in cell proliferation, survival, and migration and correlates with decreased survival in multiple solid tumors. However, a proportion of these malignancies have little or no expression of EGFR. CpG island hypermethylation and associated transcriptional silencing are common in solid tumors. The methylation status of the EGFR CpG island was examined in a series of cell lines and tissues. Dense EGFR methylation (90%) was found in the breast cancer cell line CAMA1, and a moderate degree of methylation (30-50%) was observed in the breast cancer cell lines MB435 and MB453. Transcriptional silencing of EGFR in these cell lines closely correlated with methylation. By contrast, no methylation of the HER-2/ neu CpG island was detected. EGFR hypermethylation was also found in a subset of unselected primary breast (20%), head and neck squamous cell carcinoma (35%), and lung tumors (11%). Treatment with decitabine resulted in the reexpression of EGFR in CAMA1 and MB453. Both cell lines are relatively resistant to killing by the EGFR inhibitor gefitinib. However, after cotreatment with decitabine and gefitinib, a significant effect on the induction of apoptosis was observed. In conclusion, EGFR is hypermethylated and silenced in a subset of solid tumor cell lines and primary tumor specimens, and cotreatment with decitabine and gefitinib has an additive effect only in EGFR methylated breast cancer cell lines.

Topics: Antineoplastic Agents; Azacitidine; Breast Neoplasms; Cell Line; Cell Line, Tumor; Decitabine; Dinucleoside Phosphates; DNA Methylation; DNA Primers; Epigenesis, Genetic; ErbB Receptors; Female; Gefitinib; Genetic Therapy; Humans; Organ Specificity; Quinazolines; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction

Several genes that contain the PR (PRDI-BF1 and RIZ) domain have been linked with human cancers. We describe here a new PR-domain-containing gene designated as PRDM5 (PFM2). A PRDM5 cDNA was isolated based on its homology to the PR domain of RIZ1 (PRDM2). The gene encodes an open reading frame of 630 amino acids and contains a PR domain in the NH-terminal region followed by 16 zinc finger motifs. Radiation hybrid analysis mapped PRDM5 to human chromosome 4q26, a region thought to harbor tumor suppressor genes for breast, ovarian, liver, lung, colon, and other cancers. The gene has a CpG island promoter and is silenced in human breast, ovarian, and liver cancers. A recombinant adenovirus expressing PRDM5 caused G2/M arrest and apoptosis upon infection of tumor cells. These results suggest that inactivation of PRDM5 may play a role in carcinogenesis.

Topics: Amino Acid Sequence; Apoptosis; Base Sequence; Breast; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line, Tumor; Dinucleoside Phosphates; DNA-Binding Proteins; Female; Gene Silencing; Humans; Liver; Liver Neoplasms; Molecular Sequence Data; Ovarian Neoplasms; Transcription Factors

In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions.. Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRbeta1, RARbeta2, HIC1, APC, CCND2, and CDH1 genes.. The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRbeta1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARbeta2 locus (P < 0.03).. Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

Topics: Analysis of Variance; Breast Neoplasms; Dinucleoside Phosphates; DNA Methylation; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Humans; Polymerase Chain Reaction; Promoter Regions, Genetic

ICBP90, inverted CCAAT box-binding protein of 90 kDa, has been reported as a regulator of topoisomerase IIalpha expression. We present evidence here that ICBP90 binds to methyl-CpG when at least one symmetrically methylated-CpG dinucleotides is presented as its recognition sequence. A SET and RING finger-associated (SRA) domain accounts for the high binding affinity of ICBP90 for methyl-CpG dinucleotides. This protein constitutes a complex with HDAC1 also via its SRA domain, and bound to methylated promoter regions of various tumor suppressor genes, including p16INK4Aand p14ARF, in cancer cells. It has been reported that expression of ICBP90 was upregulated by E2F-1, and we confirmed that the upregulation was caused by binding of E2F-1 to the intron1 of ICBP90, which contains two E2F-1-binding motifs. Our data also revealed accumulation of ICBP90 in breast-cancer cells, where it might suppress expression of tumor suppressor genes through deacetylation of histones after recruitment of HDAC1. The data reported here suggest that ICBP90 is involved in cell proliferation by way of methylation-mediated regulation of certain genes.

Topics: Breast Neoplasms; CCAAT-Enhancer-Binding Proteins; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cloning, Molecular; Dinucleoside Phosphates; DNA Methylation; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Genes, Tumor Suppressor; Histone Deacetylase 1; Histone Deacetylases; Humans; Kidney; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Protein Binding; Recombinant Proteins; Transcription Factors; Transfection; Ubiquitin-Protein Ligases

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Topics: Adenocarcinoma; Base Sequence; Brain Neoplasms; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Colonic Neoplasms; Dinucleoside Phosphates; DNA Methylation; Female; Genome, Human; Humans; Male; Molecular Sequence Data; Neoplasms; Restriction Mapping

We have developed methylation-sensitive, single-strand conformation analysis (MS-SSCA) as a method of screening for methylation changes. Bisulfite modification converts cytosines to thymines, but methylated cytosines remain unchanged. This modification creates sequence differences between methylated and unmethylated samples, which can be resolved by SSCA. SSCA is 70-95% efficient at detecting single base changes in a fragment. As bisulfite modification of methylated DNA would typically involve several base changes in a fragment, the efficiency of detecting methylation using MS-SSCA could approach 100%. We applied this method to analyze the BRCA1 promoter CpG island in breast cancer samples. About 20% of sporadic breast cancers are hypermethylated at the BRCA1 promoter CpG island. MS-SSCA rapidly detected those tumors that had previously been shown to be methylated by Southern blotting. The variant bands detected by SSCA were analyzed by sequencing and shown to be methylated. MS-SSCA is a simple method for screening large numbers of samples for methylation and can accelerate genomic sequencing, as all bands can be isolated and sequenced directly.

Topics: Base Sequence; BRCA1 Protein; Breast Neoplasms; Dinucleoside Phosphates; DNA Methylation; Female; Genes, BRCA1; Genetic Testing; Humans; Indicators and Reagents; Molecular Sequence Data; Polymerase Chain Reaction; Promoter Regions, Genetic; Reproducibility of Results; Sensitivity and Specificity; Sequence Alignment

Breast cancer is a genetic disease arising from a series of germ-line and/or somatic DNA changes in a variety of genes, including BRCA1 and BRCA2. DNA modifications have been shown to occur by a number of mechanisms that include DNA methylation. In some cases, the aberrant methylation of CpGs within 5' regulatory regions has led to suppression of gene activity. In this report we describe a variation in the pattern of DNA methylation within the regulatory region of the BRCA1 gene. We found no evidence of methylation at CpGs within the BRCA1 promoter in a variety of normal human tissues. However, screening of a series of randomly sampled breast carcinomas revealed the presence of CpG methylation adjacent to the BRCA1 transcription start site. One such methylated CpG occurs at a putative CREB (cAMP-responsive element binding) transcription factor binding site in the BRCA1 promoter. Gelshift assays with methylated and unmethylated BRCA1/CREB binding site oligonucleotides demonstrate that this site is sensitive to site-specific CpG methylation. These data suggest that aberrant DNA methylation at regulatory sequences in the BRCA1 locus may play a role in the transcriptional inactivation of the BRCA1 gene within subclones of breast tumors. This study represents the first evidence suggesting a role for DNA methylation in the transcriptional inactivation of the BRCA1 in human breast cancer.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Cyclic AMP Response Element-Binding Protein; Dinucleoside Phosphates; DNA Methylation; Exons; Female; Genes, BRCA1; Humans; Models, Genetic; Oligodeoxyribonucleotides; Ovarian Neoplasms; Promoter Regions, Genetic; Reference Values; Regulatory Sequences, Nucleic Acid; Transcription, Genetic

Germline mutations of the BRCA2 gene on chromosome 13q12-q13 predispose to the development of early-onset breast cancer and ovarian cancer. Loss of heterozygosity detected using chromosome 13q markers in the vicinity of BRCA2 is observed in most cancers arising in carriers of germline BRCA2 mutations and also in 30-50% of sporadic breast and ovarian cancers. However, somatic mutations of BRCA2 are extremely rare in sporadic cancers. We have examined the hypothesis that expression of the BRCA2 gene may be suppressed in sporadic breast cancers by a mechanism that is associated with increased methylation of cytosine residues in the promoter region. Using a HpaII/MspI digestion-polymerase chain reaction based assay, the presence of 5-methylcytosine in three CpG dinucleotides within the BRCA2 promoter was assessed in 18 breast or ovarian cancer cell lines, in an SV40 large T antigen immortalized cell line derived from normal breast epithelial cells, in 64 primary sporadic breast cancers and peripheral blood leucocytes from these cases and in a number of other normal human tissues. Methylation was not detected in any of the tissues examined, suggesting that this mechanism of transcriptional repression is unlikely to explain the absence of somatic mutations in sporadic cancers.

Topics: Amino Acid Sequence; BRCA2 Protein; Breast Neoplasms; Dinucleoside Phosphates; DNA Methylation; Female; Genetic Markers; Humans; Molecular Sequence Data; Neoplasm Proteins; Ovarian Neoplasms; Promoter Regions, Genetic; Transcription Factors

The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.

Topics: Adult; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cell Line; Child; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 9; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Glioma; Homozygote; Humans; Leukemia; Leukemia, Myeloid, Acute; Lung Neoplasms; Lymphocytes; Male; Methylation; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prostatic Neoplasms; Reference Values; Restriction Mapping; Tumor Cells, Cultured; Tumor Suppressor Proteins

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Alleles; Bone Marrow; Breast Neoplasms; Cell Line; Child; Child, Preschool; Colon; Colonic Neoplasms; Dinucleoside Phosphates; DNA Methylation; DNA Primers; Female; Humans; Insulin-Like Growth Factor II; Leukemia; Lung Neoplasms; Lymphocytes; Male; Middle Aged; Neoplasms; Polymerase Chain Reaction; Promoter Regions, Genetic; Tumor Cells, Cultured