Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2'-deoxycytidine treatment induces a senescence-like state.
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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)
Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction-modification system.
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In a previous study, a recombinant plasmid that contains a CACAG pentanucleotide repeat was isolated from a Pasteurella haemolytica A1 library. Southern hybridization analysis using a (CACAG)5probe indicated the presence of two loci that contain the pentanucleotide repeats on the genome of P.haemolytica A1. Additional hybridization analyses against genomic DNA from related microorganisms indicated that the repeats are only present in P.haemolytica and Pasteurella trehalosi T3. The various serotypes of P.haemolytica werefound to have either one or two of the CACAG repeat-containing loci. Examination of the locus designated Rpt2 by PCR and sequence analysis indicated that the number of CACAG repeats could change upon serial subculture which most likely occurs as a result of DNA slipped-strand mispairing. A plasmid carrying the Rpt2 locus was isolated and characterized. Sequenceanalysis indicated that the CACAG repeats are contained within the 5'-end of a gene that showed homology to mod genes of type III restriction-modification systems. A second open reading frame downstream was identified which showed homology to res genes of type III restriction-modification systems. Both the modification and restriction proteins could be expressed and polypeptides of the expected sizes were detected by SDS-PAGE. Restriction activity could also be detected in crude cytoplasmic extracts of Escherichia coli strains carrying the mod and res genes on recombinant plasmids. (+info)
DNA methyltransferase is a downstream effector of cellular transformation triggered by simian virus 40 large T antigen.
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This paper tests the hypothesis that DNA methyltransferase plays a causal role in cellular transformation induced by SV40 T antigen. We show that T antigen expression results in elevation of DNA methyltransferase (MeTase) mRNA, DNA MeTase protein levels, and global genomic DNA methylation. A T antigen mutant that has lost the ability to bind pRb does not induce DNA MeTase. This up-regulation of DNA MeTase by T antigen occurs mainly at the posttranscriptional level by altering mRNA stability. Inhibition of DNA MeTase by antisense oligonucleotide inhibitors results in inhibition of induction of cellular transformation by T antigen as determined by a transient transfection and soft agar assay. These results suggest that elevation of DNA MeTase is an essential component of the oncogenic program induced by T antigen. (+info)
Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.
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Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for gamma interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5' CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell line, was treated with gamma interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma cell line, only did so when treated with 5-aza-2'-deoxycytidine followed by gamma interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of gamma interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell-lineage lymphomas. (+info)
Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis.
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Somatic changes in CpG dinucleotide methylation occur quite commonly in human cancer cell DNA. Relative to DNA from normal human colonic cells, DNA from human colorectal cancer cells typically displays regional CpG dinucleotide hypermethylation amid global CpG dinucleotide hypomethylation. The role of the maintenance DNA methyltransferase (DNMT1) in the acquisition of such abnormal CpG dinucleotide methylation changes in colorectal cancer cells remains controversial; in one study, 60-200-fold increases in DNMT1 mRNA expression were detected in colorectal polyps and cancers relative to normal colonic tissue [W. S. El-Deiry et al., Proc. Natl. Acad. Sci. USA, 88: 3470-3474, 1991], whereas in another study, only small increases in DNMT1 mRNA expression, commensurate with differences in cell proliferation accompanying colonic tumorigenesis, were observed [P. J. Lee et al., Proc. Natl. Acad. Sci. USA, 93: 10366-10370, 1996]. To definitively ascertain whether abnormal DNMT1 expression might accompany human colorectal carcinogenesis, we subjected a series of normal and neoplastic colonic tissues to immunohistochemical staining using a polyclonal antiserum raised against a DNMT1 polypeptide. A concordance of DNMT1 expression with the expression of PCNA and other cell proliferation markers, such as Ki-67 and DNA topoisomerase IIalpha, was observed in normal colonic epithelial cells and in cells comprising other normal epithelia and lymphoid tissues. The polypeptide p21, which has been reported to undermine DNMT1 binding to proliferating cell nuclear antigen at DNA replication sites, was not expressed by normal colonic cells containing DNMT1 and other cell proliferation markers. In adenomatous polyps, although DNMT1 expression coincided with the expression of other cell proliferation markers, many DNMT1-expressing cells also expressed p21. The fidelity of DNMT1 expression was further undermined in colorectal carcinomas, in which a striking heterogeneity in DNMT1 expression, with some carcinoma cells containing very high DNMT1 levels and others containing very low DNMT1 levels, was observed. These results indicate that human colorectal carcinogenesis is accompanied by a progressive dysregulation of DNMT1 expression and suggest that abnormalities in DNMT1 expression may contribute to the abnormal CpG dinucleotide methylation changes characteristic of human colorectal carcinoma cell DNA. (+info)
Molecular characterization of a ranavirus isolated from largemouth bass Micropterus salmoides.
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An iridovirus, isolated from largemouth bass Micropterus salmoides following a die-off among adult fish and provisionally designated largemouth bass virus (LMBV), was characterized by analysis of viral protein synthesis in infected cells, viral DNA restriction fragment length polymorphisms (RFLP), and sequence determination of the major capsid protein and viral DNA methyltransferase genes. All 3 approaches yielded results consistent with the suggestion that LMBV was a member of the genus Ranavirus. Moreover, LMBV was nearly identical to 2 isolates from Southeast Asia which had been previously detected in imported ornamental fish. It remains to be determined whether infection of largemouth bass resulted from exposure to an imported virus, or whether the presence of similar viruses in southeast Asia and the southeastern United States indicates that iridovirus species are not geographically limited as suggested earlier, but rather globally distributed. (+info)
Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae.
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Several mutants ( spb1 - spb7 ) have been previously identified as cold-sensitive extragenic suppressors of loss-of-function mutations in the poly(A)(+)-binding protein 1 of Saccharomyces cerevisiae. Cloning, sequence and disruption analyses revealed that SPB1 (YCL054W) encodes an essential putative S -adenosylmethionine-dependent methyltransferase. Polysome analyses showed an under-accumulation of 60S ribosomal subunits in the spb1-1 mutant and in a strain genetically depleted of Spb1p. Northern and primer extension analyses indicated that this was due to inhibition of processing of the 27SB precursors, which results in depletion of the mature 25S and 5.8S rRNAs. At later time points of Spb1p depletion, the stability of 40S ribosomal subunits is also affected. These results suggest that Spb1p is involved in 60S ribosomal subunit biogenesis and associates early with the pre-ribosomes. Consistent with this, hemagglutinin epitope-tagged Spb1p localizes to the nucleus with nucleolar enrichment. Despite the expected methyltransferase activity of Spb1p, global methylation of pre-rRNA is not affected upon Spb1p depletion. We propose that Spb1p is required for proper assembly of pre-ribosomal particles during the biogenesis of 60S ribosomal subunits. (+info)
Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells.
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Inhibitors of DNA methyltransferase, typified by 5-aza-2'-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-alpha. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT's limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-alpha2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors. (+info)