Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer.
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Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression. (+info)
Comparative sequence analysis of human minisatellites showing meiotic repeat instability.
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The highly variable human minisatellites MS32 (D1S8), MS31A (D7S21), and CEB1 (D2S90) all show recombination-based repeat instability restricted to the germline. Mutation usually results in polar interallelic conversion or occasionally in crossovers, which, at MS32 at least, extend into DNA flanking the repeat array, defining a localized recombination hotspot and suggesting that cis-acting elements in flanking DNA can influence repeat instability. Therefore, comparative sequence analysis was performed to search for common flanking elements associated with these unstable loci. All three minisatellites are located in GC-rich DNA abundant in dispersed and tandem repetitive elements. There were no significant sequence similarities between different loci upstream of the unstable end of the repeat array. Only one of the three loci showed clear evidence for putative coding sequences near the minisatellite. No consistent patterns of thermal stability or DNA secondary structure were shared by DNA flanking these loci. This work extends previous data on the genomic environment of minisatellites. In addition, this work suggests that recombinational activity is not controlled by primary or secondary characteristics of the DNA sequence flanking the repeat array and is not obviously associated with gene promoters as seen in yeast. (+info)
Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia.
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The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the O6 position of guanine. MGMT expression is decreased in some tumor tissues, and lack of activity has been observed in some cell lines. Loss of expression is rarely due to deletion, mutation, or rearrangement of the MGMT gene, but methylation of discrete regions of the CpG island of MGMT has been associated with the silencing of the gene in cell lines. We used methylation-specific PCR to study the promoter methylation of the MGMT gene. All normal tissues and expressing cancer cell lines were unmethylated, whereas nonexpressing cancer cell lines were methylated. Among the more than 500 primary human tumors examined, MGMT hypermethylation was present in a subset of specific types of cancer. In gliomas and colorectal carcinomas, aberrant methylation was detected in 40% of the tumors, whereas in non-small cell lung carcinomas, lymphomas, and head and neck carcinomas, this alteration was found in 25% of the tumors. MGMT methylation was found rarely or not at all in other tumor types. We also analyzed MGMT expression by immunohistochemistry in relation to the methylation status in 31 primary tumors. The presence of aberrant hypermethylation was associated with loss of MGMT protein, in contrast to retention of protein in the majority of tumors without aberrant hypermethylation. Our results suggest that epigenetic inactivation of MGMT plays an important role in primary human neoplasia. (+info)
Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers.
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Tissue inhibitor of metalloproteinase-3 (TIMP-3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastasis. Loss of TIMP-3 has been related to the acquisition of tumorigenesis. Herein, we show that TIMP-3 is silenced in association with aberrant promoter-region methylation in cell lines derived from human cancers. TIMP-3 expression was restored after 5-aza-2'deoxycytidine-mediated demethylation of the TIMP-3 proximal promoter region. Genomic bisulfite sequencing revealed that TIMP-3 silencing was related to the overall density of methylation and that discrete regions within the TIMP-3 CpG island may be important for the silencing of this gene. Aberrant methylation of TIMP-3 occurred in primary cancers of the kidney, brain, colon, breast, and lung, but not in any of 41 normal tissue samples. The most frequent TIMP-3 methylation was found in renal cancers, which originate in the tissue that normally expresses the highest TIMP-3 levels. This methylation correlated with a lack of detectable TIMP-3 protein in these tumors. Together, these data show that methylation-associated inactivation of TIMP-3 is frequent in many human tumors. (+info)
Chlamydia infections and heart disease linked through antigenic mimicry.
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Chlamydia infections are epidemiologically linked to human heart disease. A peptide from the murine heart muscle-specific alpha myosin heavy chain that has sequence homology to the 60-kilodalton cysteine-rich outer membrane proteins of Chlamydia pneumoniae, C. psittaci, and C. trachomatis was shown to induce autoimmune inflammatory heart disease in mice. Injection of the homologous Chlamydia peptides into mice also induced perivascular inflammation, fibrotic changes, and blood vessel occlusion in the heart, as well as triggering T and B cell reactivity to the homologous endogenous heart muscle-specific peptide. Chlamydia DNA functioned as an adjuvant in the triggering of peptide-induced inflammatory heart disease. Infection with C. trachomatis led to the production of autoantibodies to heart muscle-specific epitopes. Thus, Chlamydia-mediated heart disease is induced by antigenic mimicry of a heart muscle-specific protein. (+info)
Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer.
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BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC. (+info)
Genomic structure and expression of the mouse growth factor receptor related to tyrosine kinases (Ryk).
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We report the genomic organization of the mouse orphan receptor related to tyrosine kinases (Ryk), a structurally unclassified member of the growth factor receptor family. The mouse RYK protein is encoded by 15 exons distributed over a minimum of 81 kilobases. Genomic DNA sequences encoding a variant protein tyrosine kinase ATP-binding motif characteristic of RYK are unexpectedly found in two separate exons. A feature of the gene is an unmethylated CpG island spanning exon 1 and flanking sequences, including a TATA box-containing putative promoter and single transcription start site. Immunohistochemical examination of RYK protein distribution revealed widespread but developmentally regulated expression, which was spatially restricted within particular adult organs. Quantitative reduction of Southern blotting stringency for the detection of Ryk-related sequences provided evidence for a retroprocessed mouse pseudogene and a more distantly related gene paralogue. Extensive cross-species reactivity of a mouse Ryk kinase subdomain probe and the cloning of a Ryk orthologue from Caenorhabditis elegans demonstrate that Ryk and its relatives encode widely conserved members of a novel receptor tyrosine kinase subfamily. (+info)
Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis.
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We have constructed a library of DNA fragments heavily methylated in human adenocarcinomas of the lung to permit the comprehensive isolation of methylated CpG islands in cancer. Heavily methylated genomic DNA fragments from tumors of nine male patients were enriched using a methylated DNA binding column and used for construction of the library. From this library, DNA fragments having properties of CpG islands were isolated on the basis of their reduced rate of strand dissociation during denaturing gradient gel electrophoresis. Approximately 1,000 clones, corresponding to 0.3% of the library were analyzed, and nine DNA fragments were identified as being associated with CpG islands that were methylated in tumor DNA. One CpG island was methylated specifically in tumor DNA, whereas the remaining eight CpG islands were methylated both in normal and tumor DNA derived from the same patients. Our results suggest that the number of CpG islands methylated specifically in tumors is not large. The library, which contains DNA fragments from methylated CpG islands comprehensively, is expected to be valuable when elucidating epigenetic processes involved in carcinogenesis. (+info)