Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation.
(41/981)
Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites. (+info)
Loss of estrogen receptor (ER) expression in endometrial tumors is not associated with de novo methylation of the 5' end of the ER gene.
(42/981)
Normal endometrium, an estrogen-responsive tissue, expresses the estrogen receptor (ER) alpha gene. Loss of ER expression, the basis for which is currently unknown, is often seen in advanced stage, poor prognosis endometrial tumors. The ER gene undergoes de novo methylation with high frequency in a wide variety of human tumors, including ER-negative breast cancers. In this study, we used several bisulfite-based detection methods to assess whether loss of ER positivity in endometrial tumors is associated with aberrant methylation of the ER gene. Although extensive methylation of a 600-bp region at the 5' end of the gene was seen in two endometrial carcinoma cell lines, none of the 55 CpGs in this region was methylated in 25 of 26 ER-deficient endometrial carcinomas. (+info)
Correlations of partial and extensive methylation at the p14(ARF) locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors.
(43/981)
p14(ARF) is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1beta but not the immediate upstream CpG sites. p14(ARF) mRNA was expressed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 microM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14(ARF) was often accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylated tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described. (+info)
Relationship between superoxide dismutase and pathogenic mechanisms of Listeria monocytogenes.
(44/981)
Listeria monocytogenes was examined for superoxide dismutase(SOD) activity. Two catalase-negative strains possessed at least twofold greater SOD activities than the catalase-positive L. monocytogenes strains examined. Growth conditions such as aeration and iron concentration influenced the specific activity of SOD obtained from cells cultured in defined media. L. monocytogenes SOD from crude extracts and after partial purification was analyzed by polyacrylamide gel electrophoresis. Iron was associated with the single band of SOD activity detected in the gels. SOD activity appeared to be primarily extracytoplasmic. Survival of organisms in a superoxide-generating medium was studied, with photoactivation of riboflavin used as the source of free radical formation. Virulent, catalase-positive L. monocytogenes strains were relatively resistant to killing in a pH 7 superoxide-containing medium. An intact-cell assay for SOD was developed, which used the superoxide-generating system and employed the superoxide-dependent oxidation of sulfite, added to the medium, and inhibition of this oxidation by SOD. Maximal SOD activites of intact cells were observed when 100 to 400 micrograms (dry weight) of viable Listeria cells per ml was added to the medium. A possible role for SOD in the pathogenesis of listeric infection is discussed. (+info)
Recombinant Arabidopsis SQD1 converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro.
(45/981)
The sulfolipid sulfoquinovosyldiacylglycerol is a component of plant photosynthetic membranes and represents one of the few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a 6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The formation of the sulfonic acid precursor, UDP-sulfoquinovose, from UDP-glucose and a sulfur donor is proposed to be catalyzed by the bacterial SQDB proteins or the orthologous plant SQD1 proteins. To investigate the underlying enzymatic mechanism and to elucidate the de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1 protein from Arabidopsis thaliana. Among different possible sulfur donors tested, sulfite led to the formation of UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1 T145A mutant showed greatly reduced activity. The UDP-sulfoquinovose formed in this assay was identified by co-chromatography with standards and served as substrate for the sulfolipid synthase associated with spinach chloroplast membranes. Approximate K(m) values of 150 microm for UDP-glucose and 10 microm for sulfite were established for SQD1. Based on our results, we propose that SQD1 catalyzes the formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived from the sulfate reduction pathway in the chloroplast. (+info)
Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma.
(46/981)
The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC. (+info)
Methionine-to-cysteine recycling in Klebsiella aerogenes.
(47/981)
In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5'-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella. (+info)
Dynamic readjustment of parental methylation patterns of the 5'-flank of the mouse H19 gene during in vitro organogenesis.
(48/981)
Gametic marks are stably propagated in order to manifest parent of origin-specific expression patterns of imprinted genes in the developing conceptus. Although the character of the imprint has not yet been fully elucidated, there is compelling evidence that it involves a methylation mark. This is exemplified by a region upstream of the H19 gene, which is not only methylated in a parent of origin-specific manner, but also regulates the silencing of the maternal Igf2 and paternal H19 alleles, respectively. We show here that the parental-specific methylation patterns within the differentially methylated domain (DMD) are perturbed in the soma during in vitro organogenesis. Under these conditions, the paternal DMD allele becomes partially demethylated, whereas the maternal DMD allele gains methylation. Despite these effects, there were no changes in allelic Igf2 or H19 expression patterns in the embryo. Finally, we show that although TSA derepresses the paternal H19 allele in ectoplacental cone when in vitro developed, there is no discernible effect on the methylation status of the paternally inherited 5'-flank in comparison to control samples. Collectively, this data demonstrates that the parental mark is sensitive to a subset of environmental cues and that a certain degree of plasticity of the gametic mark is tolerated without affecting the manifestation of the imprinted state. (+info)