Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer.
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)
Nonmethylated transposable elements and methylated genes in a chordate genome.
The genome of the invertebrate chordate Ciona intestinalis was found to be a stable mosaic of methylated and nonmethylated domains. Multiple copies of an apparently active long terminal repeat retrotransposon and a long interspersed element are nonmethylated and a large fraction of abundant short interspersed elements are also methylation free. Genes, by contrast, are predominantly methylated. These data are incompatible with the genome defense model, which proposes that DNA methylation in animals is primarily targeted to endogenous transposable elements. Cytosine methylation in this urochordate may be preferentially directed to genes. (+info)
Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element.
Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms. (+info)
Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia.
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.
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)
Frequent silencing of the GPC3 gene in ovarian cancer cell lines.
GPC3 encodes a glypican integral membrane protein and is mutated in the Simpson-Golabi-Behmel syndrome. Simpson-Golabi-Behmel syndrome, an X-linked condition, is characterized by pre- and postnatal overgrowth as well as by various other abnormalities, including increased risk of embryonal tumors. The GPC3 gene is located at Xq26, a region frequently deleted in advanced ovarian cancers. To determine whether GPC3 is a tumor suppressor in ovarian neoplasia, we studied its expression and mutational status in 13 ovarian cancer cell lines. No mutations were found in GPC3, but its expression was lost in four (31%) of the cell lines analyzed. In an of the cases where GPC3 expression was lost, the GPC3 promoter was hypermethylated, as demonstrated by Southern analysis. Expression of GPC3 was restored by treatment of the cells with the demethylating agent 5-aza-2'-deoxycytidine. A colony-forming assay confirmed that ectopic GPC3 expression inhibited the growth of ovarian cancer cell lines. Our results show that GPC3, a gene involved in the control of organ growth, is frequently inactivated in a subset of ovarian cancers and suggest that it may function as a tumor suppressor in the ovary. (+info)
Clonality of isolated eosinophils in the hypereosinophilic syndrome.
The idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII and Hha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression. (+info)
Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2'-deoxycytidine treatment induces a senescence-like state.
We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16ink4a in response to treatment with a demethylating agent. (+info)