Methylation of the c-myc gene changes during aging process of mice

The degree of methylation at c-myc proto-oncogene was found to change during aging process of mice by the use of methylation-sensitive restriction enzymes. The spleen DNA showed hypomethylation as mice aged, while hypermethylation was observed in the liver DNA. The brain DNA on the contrary revealed no appreciable difference between young and old mice. When the DNAs were examined at actin and dihydrofolate reductase (DHFR), no significant change was observed. It suggests that an age-related change of oncogene structure may be one of the factors which are related to an age-associated increase of cancer incidence rate.     

Tissue specific methylation of c-myc in adult chickens

Methylation of cytosine in DNA has long been correlated with modulation of specific gene expression in eukaryotes. Methylation of the c-myc locus was examined in six tissues from adult Leghorn chickens. The c-myc locus was found to be variably methylated in all examined tissues, except blood, where erythrocytic DNA showed no evidence of significant methylation of c-myc. This is contrasted with the observed sever methylation of the beta actin locus and the generally high methylation patterns found in avian erythrocytic DNA.     

Suppression of the EGF-dependent induction of c-myc proto-oncogene expression by transforming growth factor beta in a human breast carcinoma cell line

Alterations in c-myc proto-oncogene expression after treatment of human mammary carcinoma MDA-468 cells with epidermal growth factor (EGF) and/or transforming growth factor beta (TGF beta) have been investigated. A stimulation of c-myc messenger RNA was detected within 60 min after treatment with EGF. This induction persisted for at least 24 hr, albeit to a lower extent. The early and late increase in c-myc mRNA levels induced by EGF were inhibited by the presence of TGF beta. TGF beta alone induced little change in c-myc mRNA levels. The effect of TGF beta represents a novel action of this hormone at the level of gene expression.     

Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.     

DNA methylation at mammalian replication origins.

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.     

Folic acid and sodium butyrate prevent tumorigenesis in a mouse model of colorectal cancer

Colorectal cancer is a leading cause of morbidity and mortality worldwide, and its incidence has been increasing in recent years. The role of epigenetic modifications, including DNA methylation and histone modifications, has only recently been investigated. In this study, the effects of epigenetic agents such as folic acid (FA) and sodium butyrate (NaBu) on the development of colorectal cancer induced by 1,2-dimethylhydrazine (DMH) using ICR mice was examined. Of the mice treated in a chemopreventive manner with epigenetic agents, FA and NaBu, 15–50% developed colorectal cancer at 24 weeks compared with a 95% incidence of colorectal cancer in DMH-treated control mice. Folate deficiency can alter cytosine methylation in DNA leading to inappropriate activation of the proto-oncogene c-myc. We detected lower levels of p21WAF1 gene expression in colorectal cancer samples, as well as significantly lower levels of acetylated histone H3, compared with samples from corresponding normal colorectal mucosa. In contrast, administration of NaBu increased levels of p21WAF1 mRNA and p21WAF1 protein, and was associated with an accumulation of histone acetylation. In summary, our results show that FA and NaBu reduce the incidence of colorectal cancer induced by DMH-induced in ICR mice, and therefore we hypothesize that targeting epigenetic targets should be further investigated for the prevention of colorectal cancer in humans.     

Clonal variation in gene methylation: c-H-ras and alpha-hCG regions vary independently in human fibroblast lineages

The stability of DNA methylation has been followed in clonal lineages of human diploid fibroblasts, for the gene regions encoding the c-H-ras proto-oncogene and the alpha subunit of human chorionic gonadotropin (alpha-hCG). Although methylation losses predominated, both de novo gains and losses of cytosine methylation were observed in subclones and sub-subclones, at frequencies which differed between individual clonal lineages, and between the 2 gene regions compared. Methylation of these loci varied independently among clones; e.g., a lineage which showed frequent methylation loss in the c-H-ras gene region remained highly methylated for alpha-hCG, and vice versa. Thus, the fidelity with which DNA methylation is inherited in specific endogenous gene regions must be governed by a clone-specific property affecting local chromatin structure, but apparently not by gene expression per se. Late in the replicative life-span of diploid fibroblasts, as cell replication slowed, restriction patterns for methylation-sensitive enzymes became simpler and more discrete, while those for other enzymes did not change. This is interpreted as a consequence of 'clonal succession', in which the fastest-replicating or longest-lived clones/subclones eventually predominate in a cell population; it could also reflect a decreased rate or a non-random selection of methylation changes in late-passage cells.     

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.     

The effect of dichloroacetic acid and trichloroacetic acid on DNA methylation and cell proliferation in B6C3F1 mice

The chlorine disinfection by-products, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are carcinogenic in mouse liver. We have previously reported that DCA and TCA induced DNA hypomethylation in mouse liver. In the present study, we determined the temporal association for DNA hypomethylation and cell proliferation. Female B6C3F1 mice were administered daily doses of 500 mg/kg DCA or TCA by gavage and sacrificed at 24, 36, 48, 72, and 96 hours after the first dose. The proliferating cell nuclear antigen-labeling index in the liver was increased at 72 and 96 hours by both DCA and TCA, that is, at 72 hours the index was 1.00 +/- 0.21, 0.51 +/- 0.11, and 0.095 +/- 0.016 for DCA, TCA, and the vehicle control, respectively. The mitotic index was also significantly increased at 96 hours. The promoter region for the c-myc gene was hypomethylated only at 72 and 96 hours and not at the earlier sacrifices. Similarly, the methylation of the c-myc gene in the kidney and urinary bladder was decreased only at 72 and 96 hours. In summary, enhancement of cell proliferation and decreased methylation of the c-myc gene were first observed simultaneously at 72 hours after the start of exposure. Thus, the results support the hypothesis that DCA and TCA induce DNA hypomethylation by inducing DNA replication and preventing the methylation of the newly synthesized strands of DNA.     

Neoplastic transformation by the human gene N-myc.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.     

Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.     

Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA去甲基化对细胞衰老的影响

5-氮杂胞嘧啶（5aZaC）为DNA甲基化转移酶的抑制剂,用它处理细胞,使基因组DNA去甲基化．结果发现,经5aZaC处理后的细胞衰老进程明显加快,这从另一侧面反映了DNA甲基化与细胞衰老的关系．