Cellular DNA Methylation Research Articles

Caffeic acid, chlorogenic acid, EGCG가 유방암 세포 T-47D의 p16 유전자 DNA methylation에 미치는 영향

In the present investigation, we studied the modulating effects of caffeic acid, chlorogenic acid, and (-)-epigallocatechin-3-gallate(EGCG) on the methylation status of promoter regions of cell cycle regulator, p16, in human breast cancer T-47D cells. We demonstrated that treatment of T-47D cells with caffeic acid, chlorogenic acid, or EGCG partially inhibited the methylation status of the promoter regions of p16 genes determined by methylation-specific PCR. In contrast, unmethylated p16 genes were increased with the treatment of T-47D cells with 20 μM of caffeic acid or chlorogenic acid for 6 days. Treatment of T-47D cells with 5, 20 or 50 μM of EGCG increased the unmethylation status of p16 gene up to 100%, and the methylation-specific bands of this gene were decreased up to 50 % in a concentration-dependent manner. The finding of present study demonstrated that coffee polyphenols and EGCG have strong inhibitory effects of the cellular DNA methylation process through increased formation of S-adenosyl-homocysteine(SAH) during the catechol-O-methyltransferase (COMT)- mediated O-methylation of these dietary chemicals or an direct inhibition of the DNA methyltransferases. In conclusion, various dietary polyphenols could reverse the methylation status of p16 gene in human breast T-47D cells.

5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980's there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

Benzopyrene exposure disrupts DNA methylation and growth dynamics in breast cancer cells

Exposures to environmental carcinogens and unhealthy lifestyle choices increase the incidence of breast cancer. One such compound, benzo(a)pyrene (BaP), leads to covalent DNA modifications and the deregulation of gene expression. To date, these mechanisms of BaP-induced carcinogenesis are poorly understood, particularly in the case of breast cancer. We tested the effects of BaP exposure on cellular growth dynamics and DNA methylation in four breast cancer cell lines since disruptions in DNA methylation lead to deregulated gene expression and the loss of genomic integrity. We observed robust time- and concentration-dependent loss of proliferation, S phase and G2M accumulation and apoptosis in p53 positive MCF-7 and T47-D cells. We observed minimal responses in p53 negative HCC-1086 and MDA MB 231 cells. Furthermore, BaP increased p53 levels in both p53 positive cell lines, as well as p21 levels in MCF-7 cells, an effect that was prevented by the p53-specific inhibitor pifithrin-alpha. No changes in global levels of DNA methylation levels induced by BaP were detected by the methyl acceptor assay (MAA) in any cell line, however, methylation profiling by AIMS (amplification of intermethylated sites) analysis showed dynamic, sequence-specific hypo- and hypermethylation events in all cell lines. We also identified BaP-induced hypomethylation events at a number of genomic repeats. Our data confirm the p53-specific disruption of the cell cycle as well as the disruption of DNA methylation as a consequence of BaP treatment, thus reinforcing the link between environmental exposures, DNA methylation and breast cancer.

Methylmapper: A Method for High-Throughput, Multilocus Bisulfite Sequence Analysis and Reporting

Understanding the phenotypic contribution of epigenetic components is making DNA methylation pattern analysis more important in higher eukaryotic genomes as well as human disease. Bisulfite sequencing protocols report DNA methylation occupancy information as a positive assay output that allows methylation patterns to be elucidated from particular devel-opmental or disease states. Reported here is a new method for bisulfite sequencing project management, data analysis, and site-specific methylation test development that is designed for integration in high-throughput genomic and bioinformatics analyses.The phenotypic consequences of epigenetic alterations are being appreciated in nearly every biological system. In higher eukaryotes, DNA methylation patterns serve as a second genomic information code, an easily monitored proximate marker reflecting epigenetic cellular decisions. Most notably, in human cancer, abnormal cellular DNA methylation patterns can directly contribute to the mechanisms of tumorigenesis, most often through the induction of erroneous gene silencing (1). Recent work by many groups has demonstrated that DNA methylation abnormalities may be exploited for the development of powerful cancer diagnostic and prognostic tools (2).One tool in the arsenal of methylation monitoring is bisulfite sequencing (3). Bisulfite treatment of DNA causes the deamination of cytosine and conversion to thymine upon amplification and cloning. However, if the cytosine is methylated, it remains unchanged. In this way, subsequent sequencing of the amplified, mutagenized clones with conventional technology allows one to understand the methylation occupancy at each cytosine.Several groups have released publicly available tools for bisulfite sequencing experimental design or data analysis (4–7). Furthermore, online tools such as methBLAST (medgen.ugent.be/methblast) have become available for in silico bisulfite modification to aid in PCR primer design. However, the utility is limited in that they either only help with experimental design or with data analysis (i.e., pattern elucidation). For those that offer methylation pattern elucidation, they also require the experimenter to employ a locus-by-locus, one-gene-at-a-time approach. Given the revolution in automated sequencing and the capacity available at most genome centers, a combined design and analysis suite that affords investigators the opportunity to take advantage of a high-throughput, low-effort analysis procedure is missing. Most researchers frustratingly address this unmet need by performing each outlined step in the most labor-intensive but readily apparent manner. Typically, this involves a stepwise approach for each target, often aligning trace files by hand and manually performing the occupancy calculations. Moreover, as multiple-locus biomarkers are discovered and employed, validation of methylation patterns in a multi-locus manner from many samples will be necessary. Having an automated analysis capacity will become an even more pressing need.We have created an efficient package of PERL programs called MethylMapper that, when combined with a primer-picking program and BLASTN, simplifies the design and analysis of bisulfite experiments in a high-throughput environment. MethylMapper allows the data to self-organize to minimize mistakes and expedite simultaneous analyses of multiple loci. Furthermore, it makes data quality control as streamlined as possible. The package requires only PERL and NCBI-BLAST (www.ncbi.nlm.nih.gov/blast/download.shtml) and requires very little memory or CPU time. System requirements and CPU usage are dependent only upon the requirements of the BLASTN operation. The package is freely available for download at methylmapper.sourceforge.net.For demonstration purposes, we used MethylMapper to design primers and analyze bisulfite sequencing results for a genomic region spanning the second exon of the SLC4A3 gene on human chromosome 2 (Figure 1). Primer sequences for the analysis shown in Figure 1 are 5′-TGATTTGGGTAAGATTTTGGTTGTGAGTAG-3′ (forward) and 5′-CATCCCTAATAAACAAAACATAAAACT-3′ (reverse). Bisulfite conversion was performed with the EZ DNA Methylation Kit™ (Zymo Research, Orange, CA, USA) under the manufacturer’s recommended conditions. PCR amplification was performed under standard conditions that employed 0.2 pmol of primers, 40 ng of template in a 25-μL volume of water, to which 25 μL of FailSafe™ G 2× premix and 1 U of FailSafe Taq DNA Polymerase (EPICENTRE, Madison, WI, USA) were added. The cycling conditions employed a single 3-min incubation at 95°C, followed by 30 cycles of 95°C for 45 s, 52°C for 15 s, and 72°C for 30 s. Finally, a 10-min chase step was performed by incubating the sample at 72°C. The PCR product was purified from an agarose gel slice and cloned using the pCR2.1-TOPO TA Cloning

On the Biological Significance of DNA Methylation

This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past thirty years. The text does not attempt to give a complete and meticulous account of the many relevant and excellent reports published by many other laboratories, so it is not a review of the field in a conventional sense. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proven their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (i) the de novo methylation of integrated foreign genomes; (ii) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (iii) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (iv) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (v) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; what role has food-ingested DNA played in the elaboration of this mechanism?

The human papillomavirus-18 genome is efficiently targeted by cellular DNA methylation

Human papillomaviruses (HPVs) infect epithelia, including the simple and the squamous epithelia of the cervix, where they can cause cancer and precursor lesions. The molecular events leading from asymptomatic HPV infections to neoplasia are poorly understood. There is evidence that progression is modulated by transcriptional mechanisms that control HPV gene expression. Here, we report the frequent methylation of HPV-18 genomes in cell culture and in situ. DNA methylation is generally known to lead to transcriptional repression due to chromatin changes. We investigated two cell lines derived from cervical cancers, namely, C4-1, which contains one HPV-18 genome, and different clones of HeLa, with 50 HPV-18 genomes. By restriction cleavage, we detected strong methylation of the L1 gene and absence of methylation of parts of the long control region (LCR). A 3-kb segment of the HPV-18 genomes downstream of the oncogenes was deleted in both cell lines. Bisulfite sequencing showed that in C4-1 cells and two HeLa clones, 18 of the 19 CpG residues in the 1.2-kb terminal part of the L1 gene were methylated, whereas a third HeLa clone had only eight methylated CpG groups, indicating changes of the methylation pattern after the establishment of the HeLa cell line. In the same four clones, none of the 12 CpG residues that overlapped with the enhancer and promoter was methylated. In six HPV-18 containing cancers and five smears from asymptomatic patients, most of the CpG residues in the L1 gene were methylated. There was complete or partial methylation, respectively, of the HPV enhancer in three of the cancers, and lack of methylation in the remaining eight samples. The promoter sequences were methylated in three of the six cancers and four of the six smears, and unmethylated elsewhere. Our data show that epithelial cells efficiently target HPV-18 genomes for DNA methylation, which may affect late and early gene transcription.

PCR-screening of human esophageal and bronchial cancers reveals absence of adenoviral DNA sequences

A number of human esophageal (3) and bronchial (10) cancers have been characterized clinically and by their histopathology. These tumors have been investigated for the persistence of human adenoviral DNA sequences. The polymerase chain reaction (PCR) and Southern transfer hybridization (SBH) techniques have been applied. All analyses have consistently yielded negative results. These findings are discussed in the light of comparisons to the Ad12 hamster tumor system in which tumor cell or transformed cell revertants can lose the integrated Ad12 DNA sequences, but retain the oncogenic phenotype, when reinjected into hamsters. Ad12-transformed cells and Ad12-induced tumor cells have previously been shown to exhibit altered cellular methylation and transcription patterns. In one of the revertants, which has lost all Ad12 DNA sequences, changes in cellular DNA methylation patterns are also maintained. Since in the hamster tumor system the loss of Ad12 DNA sequences is still compatible with the oncogenic phenotype, the possibility exists that human tumors, though themselves devoid of viral DNA sequences, could have had cells as precursors which originally carried integrated adenoviral DNA sequences.

Microarray analysis of gene regulation in the Hepa1c1c7 cell line following exposure to the DNA methylation inhibitor 5-aza-2 ′-deoxycytidine and 2,3,7,8-tetrachlorodibenzo- p-dioxin

Differential expression of various genes was observed in the Hepa1c1c7 cell line following exposure to the DNA methylation inhibitor 5-aza-2 ′-deoxycytidine (AzaC) and to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). AzaC treatment generally affected genes induced by TCDD by modulating their induction levels. Induction of several genes, such as receptor (calcitonin) activity modifying protein 3 (Ramp3) by TCDD was enhanced by AzaC, although AzaC by itself was without effect. Some genes, such as frequently rearranged in advanced T-cell lymphomas (Frat1), were up-regulated by AzaC alone, with this induction being negatively affected by TCDD. Other genes were induced by AzaC, TCDD and their co-treatment. In contrast, many genes such as small proline-rich protein 1A (Sprr1a) and 2A (Sprr1a) were up-regulated by AzaC, but not significantly affected by TCDD. In addition, a group of genes was down-regulated by AzaC, TCDD and their co-treatment. These findings suggest the TCDD-dependent regulation of various genes to be influenced by cellular DNA methylation status.

Apoptosis in Plants: Specific Features of Plant Apoptotic Cells and Effect of Various Factors and Agents

Apoptosis is an integral part of plant ontogenesis; it is controlled by cellular oxidative status, phytohormones, and DNA methylation. In wheat plants apoptosis appears at early stages of development in coleoptile and initial leaf of 5- to 6-day-old seedlings. Distinct ultrastructural features of apoptosis observed are (1). compaction and vacuolization of cytoplasm in the apoptotic cell, (2). specific fragmentation of cytoplasm and appearance in the vacuole of unique single-membrane vesicles containing active organelles, (3). cessation of nuclear DNA synthesis, (4). condensation and margination of chromatin in the nucleus, (5). internucleosomal fragmentation of nuclear DNA, and (6). intensive synthesis of mitochondrial DNA in vacuolar vesicles. Peroxides, abscisic acid, ethylene releaser ethrel, and DNA methylation inhibitor 5-azacytidine induce and stimulate apoptosis. Modulation of the reactive oxygen species (ROS) level in seedling by antioxidants and peroxides results in tissue-specific changes in the target date for the appearance and the intensity of apoptosis. Antioxidant butylated hydroxytoluene (BHT) reduces the amount of ROS and prevents apoptosis in etiolated seedlings, prolongs coleoptile life span, and prevents the appearance of all apoptotic features mentioned. Besides, BHT induces large structural changes in the organization of all cellular organelles and the formation of new unusual membrane structures in the cytoplasm. BHT distorts mitosis and this results in the appearance of multiblade polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces differentiation of plastids with the formation of chloro(chromo)plasts. Therefore, ROS controlled by BHT seems to regulate mitosis, trigger apoptosis, and control plastid differentiation and the organization of various cellular structures formed by endocytoplasmic reticulum.

DNA methylation and chromatin - unraveling the tangled web.

Methylation of cytosines within the CpG dinucleotide by DNA methyltransferases is involved in regulating transcription and chromatin structure, controlling the spread of parasitic elements, maintaining genome stability in the face of vast amounts of repetitive DNA, and X chromosome inactivation. Cellular DNA methylation is highly compartmentalized over the mammalian genome and this compartmentalization is essential for embryonic development. When the complicated mechanisms that control which DNA sequences become methylated go awry, a number of inherited genetic diseases and cancer may result. Much new information has recently come to light regarding how cellular DNA methylation patterns may be established during development and maintained in somatic cells. Emerging evidence indicates that various chromatin states such as histone modifications (acetylation and methylation) and nucleosome positioning (modulated by ATP-dependent chromatin remodeling machines) determine DNA methylation patterning. Additionally, various regulatory factors interacting with the DNA methyltransferases may direct them to specific DNA sequences, regulate their enzymatic activity, and allow their use as transcriptional repressors. Continued studies of the connections between DNA methylation and chromatin structure and the DNA methyltransferase-associated proteins, will likely reveal that many, if not all, epigenetic modifications of the genome are directly connected. Such studies should also yield new insights into treating diseases involving aberrant DNA methylation.

Cellular Vitamins, DNA Methylation and Cancer Risk

The overall goal of this research is to evaluate interactions among cellular vitamin levels and global DNA hypomethylation and the impact of these variables on human cancer risk. Global DNA methylation was determined by two methods: a radiolabeled methyl incorporation (RMI) assay and an immunohistochemical assay using an antibody to 5-methylcytosine (5-MC). The RMI assay is useful for evaluating methylation of DNA in tissue samples, whereas the 5-MC assay clearly reveals DNA methylation in specific types of cells and has minimal day-to-day variability. We have observed significant interactions among cancer-protective vitamins and global DNA methylation at the level of tissues. A significant positive association was observed between global DNA methylation in buccal mucosal cells and malignant tissues, but not between global DNA methylation in peripheral leukocytes and malignant tissues of the lung. These results suggest that changes in global methylation in buccal mucosal cells may reflect changes in tissues at high risk of developing lung cancer. With the antibody technique, we have demonstrated that alterations in global DNA methylation are associated with epigenetic differences in susceptibility for development of lung cancer, which is involved in the progression of the disease. The effect of race on these relationships also is discussed. Significant associations observed between expression of epidermal growth factor receptor and global DNA methylation, as assessed by the 5-MC assay but not by the RMI assay, indicate that evaluation of global methylation and biomarkers in specific types of cells may shed light on the associations between global DNA methylation and other intermediate endpoint biomarkers in the future.

The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.

The latent membrane protein (LMP1) of Epstein-Barr virus (EBV) is expressed in EBV-associated nasopharyngeal carcinoma, which is notoriously metastatic. Although it is established that LMP1 represses E-cadherin expression and enhances the invasive ability of carcinoma cells, the mechanism underlying this repression remains to be elucidated. In this study, we demonstrate that LMP1 induces the expression and activity of the DNA methyltransferases 1, 3a, and 3b, using real-time reverse transcription-PCR and enzyme activity assay. This results in hypermethylation of the E-cadherin promoter and down-regulation of E-cadherin gene expression, as revealed by methylation-specific PCR, real-time reverse transcription-PCR and Western blotting data. The DNA methyltransferase inhibitor, 5'-Aza-2'dC, restores E-cadherin promoter activity and protein expression in LMP1-expressing cells, which in turn blocks cell migration ability, as demonstrated by the Transwell cell migration assay. Our findings suggest that LMP1 down-regulates E-cadherin gene expression and induces cell migration activity by using cellular DNA methylation machinery.

Foreign DNA Integration: GENOME-WIDE PERTURBATIONS OF METHYLATION AND TRANSCRIPTION IN THE RECIPIENT GENOMES

In hamster cells transgenic for the DNA of adenovirus type 12 (Ad12) or for the DNA of bacteriophage lambda, the patterns of DNA methylation in specific cellular genes or DNA segments remote from the site of transgene insertion were altered. In the present report, a wide scope of cellular DNA segments and genes was analyzed. The technique of methylation-sensitive representational difference analysis (MS-RDA) was based on a subtractive hybridization protocol after selecting against DNA segments that were heavily methylated and hence rarely cleaved by the methylation-sensitive endonuclease HpaII. The MS-RDA protocol led to the isolation of several cellular DNA segments that were indeed more heavily methylated in lambda DNA-transgenic hamster cell lines. By applying the suppressive subtractive hybridization technique to cDNA preparations from nontransgenic and Ad12-transformed or lambda DNA-transgenic hamster cells, several cellular genes with altered transcription patterns were cloned from Ad12-transformed or lambda DNA-transgenic hamster cells. Many of the DNA segments with altered methylation, which were isolated by a newly developed methylation-sensitive amplicon subtraction protocol, and cDNA fragments derived from genes with altered transcription patterns were identified by their nucleotide sequences. In control experiments, no differences in gene expression or DNA methylation patterns were detectable among individual nontransgenic BHK21 cell clones. In one mouse line transgenic for the DNA of bacteriophage lambda, hypermethylation was observed in the imprinted Igf2r gene in DNA from heart muscle. Two mouse lines transgenic for an adenovirus promoter-indicator gene construct showed hypomethylation in the interleukin 10 and Igf2r loci. We conclude that the insertion of foreign DNA into an established mammalian genome can lead to alterations in cellular DNA methylation and transcription patterns. It is conceivable that the genes and DNA segments affected by these alterations depend on the site(s) of foreign DNA insertion.

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells.

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G(0)/G(1)while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G(0)/G(1)arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.

DNA methylation of helper virus increases genetic instability of retroviral vector producer cells.

Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 x 10(5) CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 x 10(5) CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5' long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.

Integration of foreign DNA and its consequences in mammalian systems

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.

Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.

Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?

Effects of caloric restriction in animals on cellular function, oncogene expression, and DNA methylation in vitro.

While the life-extending and disease-modulating effects of caloric restriction (CR) are well documented in whole animal studies and in correlative experiments using cells taken from CR animals, very few studies have used cells in culture after their removal from the CR-fed animal. In using this in vivo-->in vitro approach we have attempted to examine the proposition that the effects of CR can be transferred to individual cells by analyzing the cellular functions of proliferation and transformation, the activation of oncogenes, and the methylation of DNA as a function only of diet. Pancreatic acinar cells excised from CR-fed Brown-Norway rats and placed in rich medium showed different responses compared to cells from ad libitum (AL)-fed controls. CR had the effect of slowing growth rate and protecting against spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation over 14 passages of cells in culture. At the molecular level, cells from the CR animals showed reduced c-Ha-ras oncogene expression and mutation as well as reduced mutation of the p53 suppressor gene. CR also increased genomic methylation of ras DNA. We conclude that the effects of CR treatment of the animal are transferred to individual cells and note that these responses (decreased proliferation and transformation; depressed oncogene expression and mutation and decreased suppressor gene mutation; and increased oncogene methylation) are cellular and molecular analogs of in vivo weight loss, life extension, and carcinogenesis modulation, which are hallmarks of CR in the whole animal. The fact that these responses are seen generations after the cells are removed from the CR-treated animal indicates that CR causes a permanent predisposition of pancreatic acinar cells to these modulated responses and shows the value of the in vivo-->in vitro protocol in studies that relate diet to cellular and molecular function.

S-Adenosylmethionine metabolism in herpes simplex virus type 2-infected cells

Infection of primary rat embryo cells with herpes simplex virus type 2 has previously been reported to produce a dramatic and rapid inhibition of cellular DNA methylation. However, it has neither an immediate effect on S-adenosylmethionine breakdown nor on the relative pool sizes of S-adenosylmethionine and S-adenosylhomocysteine.

Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis: Methylation of intact chromosomes in agarose by methylases

Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.