5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.     

5-Azacytidine decreases fragmentation of nuclear DNA and pigment formation in first leaf cells of barley seedlings

Realization of programmed cell death in senescence represents an activation/inactivation of the respective gene. Enzymatic methylation of nuclear DNA with the creation of 5methylcytosine is one of the mechanisms, which can regulate gene activity in animal and plant cells. 5Azacytidine (5azaC) acts as an inhibitor of DNA methylation, and induces expression of a range of some genes including genes responsible for senescence. Fragmentation of nuclear DNA is one of the hallmarks of programmed cell death in apoptosis pathway in plant cells. The influence of 5azaC (100 microg/ml) on nuclear DNS amount and its fragmentation in the first leaf cells of barley was studied. It was shown that in the first leaf cells of barley seedlings there is an apoptosis pathway of programmed cell death. It was also observed that nuclear DNA fragmentation under the 5azaC influence is strongly inhibited, and the DNA amount in the first leaf increases. Synthesis and destruction of chlorophyll also play a significant role in programmed cell death in plants. It was shown that under the 5azaC influence, the absorption spectrum of a pigment does not change in leaves and coleoptiles in the light, whereas in the dark condition, these pigments are not created under the 5azaC influence.     

Strong effects of 5-azacytidine on the in vitro lifespan of human diploid fibroblasts

Azacytidine (5-aza-CR) and azadeoxycytidine (5-aza-CdR) are known to inhibit the methylation of cytosine (5-mC) in DNA, and their effects on the long-term growth of human fibroblasts, strain MRC-5, have been examined. A single treatment with either analogue initially inhibits growth, but the cells recover to normal morphology, growth rate and cell density at confluence. However, a memory of the treatment is retained, since the cells' subsequent lifespan is considerably reduced in comparison with controls, and the terminal stages of growth are indistinguishable from senescent cultures of untreated cells. The effect of 5-aza-CR or 5-aza-CdR does not appear to be closely related to the concentration used, or to the length of treatment up to about half-way through the total lifespan. Sequential doses have cumulative effects on longevity. There is evidence that the pattern of 5-mC in mammalian DNA is inherited via cell division; therefore, a reduction in 5-mC induced by a pulse treatment of 5-aza-CR or 5-aza-CdR will be transmitted to all descendants. The results are consistent with independent observations that the level of 5-mC declines continually during the serial subculture of human diploid cells. The analogues would be expected to precipitate this decline and thereby advance the physiological age of the culture. The results provide support for the view that the random loss of methyl groups in DNA may eventually have deleterious consequences, such as aberrant epigenetic changes in gene expression.     

DNA damage by 5-nitro-2-furylacrylic acid, a nitrofuran derivative

5-Nitro-2-furylacrylic acid (5-NFA) caused dose dependent inhibition of growth of Escherichia coli K-12 strain AB 2480 (uvr-, rec-), the 37% (D37) and 10% (D10) survival doses being 1.0 microgram/ml.h and 1.75 micrograms/ml.h, respectively. Although much higher doses of drug were required to achieve comparable inhibition of growth of E. coli strain 1157 (repair proficient), significant filamentation of these cells was produced by treatment with 1.0 microgram/ml 5-NFA for 4 hr. Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that 5-NFA treatment of E. coli strain AB 2480 produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the initial part of the reaction obeyed a first order relation. 5-NFA also produced dose-dependent increase of prophage induction in E. coli strain GY 5027: envA, uvrB, ampA1, strA (lambda). The implications of the action of 5-NFA on DNA in relation to the induction of 'SOS' functions and carcinogenesis were discussed.     

Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.     

Stabilization of cellular properties and differentiation mutilpotential of human mesenchymal stem cells transduced with hTERT gene in a long-term culture

Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. The life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction can prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. We established a line of hMSCs transduced with exogenous hTERT (hTERT-hMSCs) and investigated its sustaining cellular properties in a long-term culture. This line of hTERT-hMSCs was cultured for 290 population doublings (PDs) without loss of contact inhibition. Under adipogenic, chondrogenic and osteogenic induction, hTERT-hMSCs at PD 95 and PD 275 could differentiate respectively into adipocytes, chondrocytes, and osteocytes. hTERT-hMSCs at these PDs showed no transforming activity through both in vitro assay of cell growth in soft agar and in vivo assay of tumorigenicity in NOD-SCID mice. Karyotype analyses showed no significant chromosomal abnormalities in hTERT-hMSCs at these PDs. These results suggested that the hTERT-hMSCs at lower population doubling levels (PDLs) should be considered as a cell model for studies of cellular senescence, differentiation and in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties.     

Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer

Both 5-methylcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) have been proposed to be involved in tumorigenesis. Because the readout of the broadly used 5mC mapping method, bisulfite sequencing (BS-seq), is the sum of 5mC and 5hmC levels, the 5mC/5hmC patterns and relationship of these two modifications remain poorly understood. By profiling real 5mC (BS-seq corrected by Tet-assisted BS-seq, TAB-seq) and 5hmC (TAB-seq) levels simultaneously at single-nucleotide resolution, we here demonstrate that there is no global loss of 5mC in kidney tumors compared with matched normal tissues. Conversely, 5hmC was globally lost in virtually all kidney tumor tissues. The 5hmC level in tumor tissues is an independent prognostic marker for kidney cancer, with lower levels of 5hmC associated with shorter overall survival. Furthermore, we demonstrated that loss of 5hmC is linked to hypermethylation in tumors compared with matched normal tissues, particularly in gene body regions. Strikingly, gene body hypermethylation was significantly associated with silencing of the tumor-related genes. Downregulation of IDH1 was identified as a mechanism underlying 5hmC loss in kidney cancer. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model. Collectively, our results demonstrate that loss of 5hmC is both a prognostic marker and an oncogenic event in kidney cancer by remodeling the DNA methylation pattern.     

Unusual effects of 5a,6-anhydrotetracycline and other tetracyclines. Inhibition of guanosine 5'-diphosphate 3'-diphosphate metabolism, RNA accumulation and other growth-related processes in Escherichia coli.

5a,6-Anhydrotetracycline was discovered to be unique among several tetracycline derivatives tested in its ability to inhibit RNA accumulation in vivo at low concentration (20 microgram/ml and less). In addition, in vivo protein, DNA, and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthesis were completely inhibited by 20 microgram/ml 5a,6-anhydrotetracycline. ppGpp decay in a spoT strain was inhibited by 20 microgram/ml 5a,6-anhydrotef RNA synthesis by a 5a,6-anhydrotetracycline may be due, in part, to reduced UTP and CTP synthesis. The effects of tetracyclines on in vitro ppGpp synthesis by crude stringent factor in the absence of ribosomes were investigated. It was determined that of six tetracyclines tested, four strongly inhibited the reaction (oxytetracycline, chlorotetracycline, dedimethylaminotetracycline, and tetracycline) whereas 5a,6-anhydrotetracycline gave a moderate inhibition and alpha-6-deoxyoxytetracycline resulted in only a slight reduction in ppGpp synthesis. It is proposed that tetracyclines interfere with factors involved in ppGpp metabolism and function.     

ING1 and 5-Azacytidine Act Synergistically to Block Breast Cancer Cell Growth

Background

Inhibitor of Growth (ING) proteins are epigenetic “readers” that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin.

Methods and Principal Findings

Here we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo.

Conclusions

These data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.     

A re-examination of the effects of ionizing radiation on lifespan and transformation of human diploid fibroblasts.

Human diploid fibroblasts, strain MRC-5, were sequentially irradiated with 60Co gamma rays at intervals during their in vitro lifespan. The results indicate that 3 or 6 doses of 1 Gy can increase lifespan, and the same was true for cells treated with 3 doses of 3 Gy. Higher doses (5 x 3 Gy) did reduce growth potential, suggesting either that mid-late passage cells become more sensitive to radiation, or that doses beyond a given threshold reduce population lifespan by multiple cellular hits. The life extension induced by gamma rays might be due to an induced hypermethylation of DNA. Alternatively, oxygen radicals produced by irradiation might trigger an adaptive stress response which would remove damaged macromolecules and thereby increase the cells' growth potential. Whichever explanation is correct, the results show that the human fibroblast system is not appropriate for the study of the well known effect of ionizing radiation in shortening the lifespan of experimental animals. Contrary to earlier published results, populations of cells treated with cumulative doses of 15 Gy or 18 Gy and held for nearly 3 months after they had reached senescence (Phase III), produced no foci of transformed cells.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

DNA甲基化作用与鼠肝细胞的老化

本文比较了不同年龄的鼠肝ＤＮＡ甲基化酶活力及ＤＮＡ甲基化水平,发现它们均与鼠龄呈反相关。又以不同年龄的鼠肝ＤＮＡ为模板,检验了其体外转录活力,发现其与鼠龄呈正相关。     

DNA methylation and cellular ageing.

Methylated cytosine (m5C) in DNA appears to be an important modulator of the expression of some genes. There are several lines of evidence that gradual loss of m5C is relevant to in vitro cellular ageing: m5C loss occurs during cell culture; m5C loss is detectable at an early stage of culture; m5C loss appears to be related to cell division not just duration in culture; the rate of m5C loss appears to be related to in vitro lifespan of the cell strain in question; and the total loss of m5C during an in vitro lifespan is significant by comparison with induced-changes in m5C levels which effect cell growth, or cause cell-death in culture. Progressive loss of m5C in dividing cells may thus produce the multi-step cell division "clock" which underlies the Hayflick phenomenon.     

Reversal of DNA methylation with 5-azacytidine alters chromosome replication patterns in human lymphocyte and fibroblast cultures

Prior studies demonstrated that developmental or induced methylation of DNA can inactivate associated gene loci. Such DNA methylation can be reversed and specific genes reactivated by treatment with 5-azacytidine (5- azaC ). The present cytogenetic studies using replication banding methods show that 5- azaC treatment also results in an increase or decrease in replication staining at one or more band locations in human lymphocyte and fibroblast chromosomes. New replication band locations are not formed. These changes in replication staining, which reflect changes in timing of replication, are different between these two tissues. However, in both tissues, the delayed onset of replication in the heterocyclic, inactive X is shortened by 5- azaC . A correlation is thus suggested between the induced temporal change to earlier DNA replication, and induced hypomethylation and gene activation. The temporal effect on chromosome replication in 5- azaC -treated cells depends on the portion of the S-period studied. Toward the beginning of S, early-replication patterns are increased in both lymphocytes and fibroblasts. Toward the end of S, late-replication patterns are increased only in lymphocytes, suggesting a differential effect of 5- azaC in: (1) early-vs. late-S, and (2) lymphocytes vs. fibroblasts. Generally, 5- azaC has its greatest effect on the inactive chromosome regions that are typically late-replicating prior to 5- azaC treatment. These observed changes in replication band staining suggest that DNA methylation may modify regional groups of genes in concert.     

Differences in sensitivity of T3, T7, T4 and lambda phages to bleomycin and phleomycin]

In contrast to phage lambda the phages T3, T7 and T4 are not inhibited by as much as 150 microgram bleomycin/ml, while the chemically related antibiotic phleomycin increasingly inhibits the propagation of the phages in the order T4-T3-lambda. 20 microgram phleomycin/ml inhibit T3 by 95%. The resistance against bleomycin is surprising, because 10 microgram BM/ml block completely the colony-forming capacity of the host bacterium. The drug resistance of the phage growth correlates with the weak decrease of phage DNA synthesis, while the host cell DNA synthesis ceases rapidly. In accordance with these data is the in vivo inhibition of Escherichia coli cells and the in vitro degradation of their DNA. However, a contradiction exists between the in vivo resistance of T3 and T4 and the in vitro susceptibility of their DNA against nucleolytical fragmentation by bleomycin. The mechanism of the insensitivity of T3, T7 and T4 against bleomycin is unknown.     

The role of 5-hydroxymethylcytosine in human cancer

The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.     

Tet family proteins and 5-hydroxymethylcytosine in development and disease

Over the past few decades, DNA methylation at the 5-position of cytosine (5-methylcytosine, 5mC) has emerged as an important epigenetic modification that plays essential roles in development, aging and disease. However, the mechanisms controlling 5mC dynamics remain elusive. Recent studies have shown that ten-eleven translocation (Tet) proteins can catalyze 5mC oxidation and generate 5mC derivatives, including 5-hydroxymethylcytosine (5hmC). The exciting discovery of these novel 5mC derivatives has begun to shed light on the dynamic nature of 5mC, and emerging evidence has shown that Tet family proteins and 5hmC are involved in normal development as well as in many diseases. In this Primer we provide an overview of the role of Tet family proteins and 5hmC in development and cancer.     

Adenovirus DNA synthesis in vitro in an isolated complex.

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.