cytidylyl-3--5--guanosine and Carcinoma--Hepatocellular

The gene encoding the tumour suppressor protein p53 is one of the most commonly mutated genes in human cancers. Analysis of the mutational events that target the p53 gene has revealed evidence for both exogenous and endogenous mutational mechanisms. For example, the p53 mutational spectrum reveals evidence for a direct causal effect of ultraviolet radiation in skin cancer, of aflatoxin B1 in liver cancer and of tobacco smoke in lung cancer. This novel field, molecular epidemiology of human cancer risk, has added a new dimension to classical associative epidemiology by providing a direct link between human cancer and carcinogen exposure.

Topics: Aflatoxin B1; Animals; Carcinogens; Carcinoma, Hepatocellular; Dinucleoside Phosphates; Genes, p53; Genes, Tumor Suppressor; Genetic Markers; Genome; Humans; Lung Neoplasms; Molecular Epidemiology; Mutation; Skin Neoplasms; Smoking; Ultraviolet Rays

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

Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylated MT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanning MT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Dinucleoside Phosphates; DNA Footprinting; DNA Methylation; DNA Modification Methylases; DNA-Binding Proteins; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Liver; Liver Neoplasms; Metallothionein; Metals, Heavy; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Rats; Sp1 Transcription Factor; Transcription Factor MTF-1; Transcription Factors

To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5' guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [(3)H]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome. BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [(3)H]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The "cytosine-extension assay" has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA.

Topics: Carcinoma, Hepatocellular; Deoxycytosine Nucleotides; Dinucleoside Phosphates; DNA; DNA Methylation; DNA Restriction Enzymes; DNA, Neoplasm; Humans; Liver Neoplasms; Restriction Mapping; Sensitivity and Specificity; Tritium; Tumor Cells, Cultured

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Nucleus; Dinucleoside Phosphates; DNA Methylation; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, Reporter; HeLa Cells; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; L Cells; Leukemia; Liver; Liver Neoplasms; Luciferases; Mice; Neuroblastoma; Nuclear Proteins; Organ Specificity; Recombinant Fusion Proteins; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Our study was designed to clarify the significance of silencing the E-cadherin gene, which is located on 16q22.1, due to CpG methylation during hepatocarcinogenesis. The CpG methylation status of primary hepatocellular carcinomas (HCCs) and corresponding liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, were assessed by digesting DNA with methylation-sensitive and non-sensitive restriction enzymes. CpG methylation around the promoter region of the E-cadherin gene was detected in 46% of liver tissues showing chronic hepatitis or cirrhosis and 67% of HCCs examined. Immunohistochemical examination revealed reduced E-cadherin expression in 59% of HCCs examined. CpG methylation around the promoter region correlated significantly with reduced E-cadherin expression in HCCs (p < 0.05). CpG methylation around the promoter region, which increases during the progression from a precancerous condition to HCC, may participate in hepatocarcinogenesis through reduction of E-cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue morphology.

Topics: Cadherins; Carcinoma, Hepatocellular; Dinucleoside Phosphates; DNA Methylation; DNA Restriction Enzymes; DNA, Neoplasm; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Precancerous Conditions; Promoter Regions, Genetic