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PubMed2003

Chromosomal aberrations induced by 5-azacytidine combined with VP-16 (etoposide) in CHO-K1 and XRS-5 cell lines.

A cytogenetic study was carried out with 5-azacytidine (5-azaC) and etoposide (VP-16) in CHO-K1 and XRS-5 (mutant cells deficient for double-strand break rejoining) cell lines to verify the interaction effects of the drugs in terms of induction of chromosomal aberrations. 5-azaC is incorporated into DNA causing DNA hypomethylation, and VP-16 (inhibitor of topoisomerase II enzyme) is a potent clastogenic agent. Cells in exponential growth were treated with 5-azaC for 1 h, following incubation for 7 h, and posttreatment with VP16 for the last 3 h. In K1 cells, the combined treatments induced a significant reduction in the aberrations induced in the X and "A" (autosome) chromosomes, which are the main target for 5-azaC. However, in XRS-5 cells, the drug combination caused a significant increase in the aberrations induced in those chromosomes, but with a concomitant reduction in the randomly induced-aberrations. In addition, each cell line presented characteristic cell cycle kinetics; while the combined treatment induced an S-arrest in K1 cells, alterations in cell cycle progression were not found for XRS-5, although each drug alone caused a G2-arrest. The different cell responses presented by the cell lines may be explained on the basis of the evidence that alterations in chromatin structure caused by 5-aza-C probably occur to a different extent in K1 and XRS-5 cells, since the mutant cells present a typical hyper-condensed chromosome structure (especially the X- and "A" chromosomes), but, alternatively, 5-aza-C could induce reactivation of DNA repair genes in XRS-5 cells.

Animals↗
PubMed1990

Suppressive effects of two bioresponse modifiers, krestin and levamisole, on 5-azacytidine-induced digital defects in rats.

The effects of two bioresponse modifiers, krestin (PSK) and levamisole hydrochloride (levamisole), on 5-azacytidine (5-AC)-induced syndactyly, brachydactyly, and ectrodactyly were investigated. Both PSK and levamisole suppressed 5-AC-induced digital malformation in the rat. The effect of PSK was significant when given 24 h before to 1 h after 5-AC treatment, and levamisole when given 12 h before to 3 h after treatment. Both agents showed an immune-stimulating effect and have been used as cancer chemotherapeutic drugs. However, as the contribution of fetal or maternal immune functions or the alleviation action is unknown, further investigations are required to clarify the mechanism.

Animals↗
PubMed1991

Hyperploid cells in the limb buds of rats and 5-azacytidine-induced digital deformities.

To clarify the mechanism underlying the digital teratogenesis of 5-azacytidine (5-AC), the DNA contents of mesenchymal cells were examined in the limb buds of the rat by microspectrophotometry. 5-AC induced hyperploid cells: 2.4% at 18 h after injection and 3.2% at 24 h. No hyperploid cells were noted 30 h after the treatment. Because posttreatment caffeine suppressed 5-AC-induced digital defects, the actions of caffeine on 5-AC-induced hyperploid cells were further studied; no hyperploid cells were detected at any sampling point. Caffeine suppressed hyperploid cells as well as digital defects. These results suggest that the causative factors of digital defects by 5-AC may involve hyperploid cells. However, in that there were too few hyperploid cells to cause the defects, further studies are required to clarify the nature of the action of 5-AC.

Abnormalities, Drug-Induced↗
PubMed2002

5-Aza-2'-deoxycytidine-induced cytotoxicity and limb reduction defects in the mouse.

BACKGROUND: 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS: Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS: Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS: These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.

Animals↗
PubMed1979

Different teratogenic efficacy to mouse fetal CNS of 5-azacytidine in combination with X-irradiation depends on the sequence of successive application.

The single treatment of pregnant mice on day 12 post conception with 5-azacytidine (AzaCr), followed by a single irradiation dose of 200 rad two hours later, is exclusively neurotoxic to the fetus, as shown by a severe hypoplasia of the parieto-occipital regions of the telencephalon. This effect is explicable by the specific function of the mitotic cell population for the integrity of the cortex wall. Combining these two hazards in the reverse manner, i.e., irradiation followed by AzaCr, resulted in no general hypoplastic effect in the forebrain and only caused a depletion of cells in the marginal cortex. This indicates a significantly diminished AzaCr sensitivity of fetal cortical cells subsequent to X-irradiation. In addition, rosette-like cell clustering in the cortex of all X-irradiated animals occurs to a similar degree, irrespective of any additional AzaCr-treatment. The only conformity between these different schedules is that a great portion of the surviving cells is most likely in the DNA synthesizing phase at the time of irradiation. It is therefore concluded that rosette formation starts perferentially from cells injured during the S-phase.

Abnormalities, Drug-Induced↗
PubMed1979

Behavioral consequences of interference with CNS development in the early fetal period.

As a part of a series of investigations into the structural and functional consequences of interference with cell proliferation, mice were treated with 5-azacytidine during two stages of early fetal life. Treatment on either the twelfth or fourteenth day of gestation led to permanent significant reductions in body weight and brain weight. Behaviorally, the earlier treatment was associated with a delay in development of the righting reflex, permanent deficits in locomotor coordination, and hypoactivity. Treatment on the fourteenth day of gestation led to decreased passive avoidance, increased active avoidance, and hyperactivity--the same syndrome observed after treatment on the eighteenth day. Both treatments led to abnormal behavior on a spatial maze task. The developmental outcome of injuries induced in the early fetal period appears similar to the outcome of injuries at later stages of development.

Animals↗
PubMed1987

Inhibitory effect of caffeine on 5-azacytidine-induced digital malformations in the rat.

The effect of caffeine on 5-azacytidine (5-AC)-induced digital malformations in rat fetuses was investigated. Caffeine suppressed all types of digital defects in the fore- and hindlimbs except for syndactyly induced by 1.0 mg/kg of 5-AC; it was still effective when administered 24 hours after 5-AC treatment. However, fetal mortality increased as the frequency of malformations decreased. While the malformation results support the view that caffeine inhibits the processes leading to malformation expression, the relation between its suppressive effect on malformations and its enhancing effect on fetal mortality is unclear.

Abnormalities, Drug-Induced↗
PubMed1989

Histological study of cell death in digital malformations induced by 5-azacytidine: suppressive effect of caffeine.

The present study investigated microscopically the process of 5-azacytidine (5-AC)-induced digital teratogenesis and caffeine's suppressive effect on this process. Three distinct zones of programmed cell death were observed in control and caffeine-treated embryos 3 hours after 5-AC injection: the preaxial and postaxial ectodermal regions and the central part of the mesodermal regions. 5-AC temporarily suppressed programmed cell death in the ectoderm and mesoderm 3 hours after it was injected. However, caffeine promoted programmed cell death; normal programmed cell death was observed in the limb buds of embryos whose dams were treated with 5-AC and caffeine. The percentage of total cell death in hindlimb buds of embryos treated with 5-AC and caffeine was higher than that from embryos treated with 5-AC, whereas 5-AC-induced digital malformations were reduced by post-treatment with caffeine. Cell death reached a maximum 12 hours after the injection in limb buds from 5-AC and caffeine-treated embryos and at 24 hours in the 5-AC treated embryos. Furthermore, in the 5-AC and caffeine-treated embryos, the frequency of cell deaths at 12 hours increased almost linearly with the doses of caffeine in parallel with the reduction of 5-AC-induced malformation frequency by caffeine. These results suggest that although induced cell death may be one of the factors leading to digital malformations produced by 5-AC, it is not essential, and the existence of other factors affecting the pattern formation of the limb bud is proposed.

Abnormalities, Drug-Induced↗
PubMed1990

Comparison of the incidence of 5-azacytidine-induced exencephaly between MT/HokIdr and Slc:ICR mice.

The incidence of 5-azacytidine-induced exencephaly was compared between MT/HokIdr strain (MT) and Slc:ICR strain (ICR) mice. MT mice have a genetic predisposition for exencephaly, but ICR mice do not. Pregnant mice were given 5-azacytidine (1 mg/kg to 100 micrograms/kg) injected intraperitoneally on Day 7.5 of gestation (vaginal plug day = Day 0.5), and fetuses were observed for external malformations on Day 18.5 of gestation. One hundred micrograms/kg 5-azacytidine induced exencephaly in MT mice but not in ICR mice, and 1 mg/kg 5-azacytidine resulted in resorptions in MT mice but caused exencephaly in ICR mice. These results indicated that MT mice had 10-fold more sensitivity to 5-azacytidine than ICR mice. It seems likely that less than effective doses of teratogens for animals without genetic predispositions are still effective in inducing malformations in animals with a genetic predisposition for malformations. When 4-somite-stage embryos of both MT and ICR mice were cultured in rat serum supplemented with 5-azacytidine, 0.02 micrograms/ml 5-azacytidine induced the failure of closure of cephalic neural tube in MT embryos but not in ICR embryos, and 0.2 micrograms/ml 5-azacytidine induced severe growth retardation in MT embryos but in ICR embryos it only induced embryos with smaller heads and fewer somites than in control. These results indicated that MT mouse embryos in culture also had a 10-fold-increased sensitivity to 5-azacytidine compared with ICR mouse embryos, suggesting maternal effects play no significant role in their increased sensitivity to 5-azacytidine.

Anencephaly↗
PubMed1990

Inhibitory effects of cobalt chloride and cinnamaldehyde on 5-azacytidine-induced digital malformations in rats.

The effect of two DNA repair inhibitors in bacteria, cobalt chloride and cinnamaldehyde, on 5-azacytidine (5-AC)-induced digital malformations was studied. Both agents inhibited the induced digital malformations. The effect of cobalt chloride was significant 3 hr before to 1 hr after the 5-AC treatment, and the effect of cinnamaldehyde was significant 3 hr before to 24 hr after the treatment. However, an increase in fetal mortality was observed with the latter agent. The mechanisms underlying the suppressive effects of both agents may be different, but their natures require elucidation.

Abnormalities, Drug-Induced↗
PubMed1993

Utility of the murine erythroleukemic cell (MELC) in assessing mechanisms of action of DNA-active developmental toxicants: application to 5-fluorouracil.

Murine erythroleukemic cells (MELC) exposed to 2'-deoxy-5-azacytidine (D-AZA) or to the active cyclophosphamide (CP) metabolites phosphoramide mustard (PAM) and 4-hydroxycyclophosphamide (OHCP) exhibit cell-cycle perturbations similar to those seen in limb bud nuclei of gestational day (GD) 10 CD-1 mouse embryos exposed in utero to D-AZA or CP, respectively. The similarities in response suggest MELC may be a useful model for determining mechanisms of action of DNA-active developmental toxicants. As such, we used the MELC model to investigate the mechanism of action of 5-fluorouracil (5-FU), an antimetabolite that induced in GD 14 rat fetuses an apparent S-phase accumulation in limb cells 8 hr after in utero exposure, but S-phase depletion in liver cells 24 hr postexposure. MELC timed-recovery and synchronization studies suggest that in proliferative tissues, 5-FU induces an early S-phase accumulation, followed by a synchronous, concentration-dependent delay in progression through the cell cycle. Consequently, it is the tissue-specific rate of delay, rather than different mechanisms of action, that results in apparent tissue-specific perturbations. Moreover, growth and cell-cycle data suggest that cells entering S phase (when TS activity is greatest) are the most sensitive to 5-FU toxicity. Assays of the TS activity of recovering MELC reveal that although the initial extent of TS inhibition does not appear to be concentration-dependent, the time to recovery is, suggesting that the rate of S-phase progression is closely associated with TS activity. Together, the induction of similar cell-cycle perturbations in embryonic/fetal tissues and MELC following exposure to CP (or CP metabolites), D-AZA, or 5-FU, as well as the adaptability of MELC to a variety of kinetic assays suggests that, for those developmental toxicants suspected of inducing cell-cycle perturbations in embryonic/fetal tissues, MELC may prove useful for elucidating mechanisms of action.

Animals↗
PubMed1994

Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2'-deoxycytidine.

DNA methylation is a probable mechanism for regulating gene expression, and alterations in methylation may significantly affect embryonic development. We administered the cytidine analogue 5-aza-2'-deoxycytidine (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death and cell cycle. Groups of females were dosed intraperitoneally on gestation day 10 with doses of 0.05-3 mg/kg dAZA and killed at 4, 8, or 28 hr later. Two embryos per litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death; the remaining embryos were frozen and stored for subsequent flow cytometric (FCM) analysis of the cellular DNA synthetic cycle in limb buds. A dose-related accumulation of cells in the S and G2/M phases was observed at 4 and 8 hr after maternal dosing. S-phase accumulation was the most sensitive indicator of effect; a dose-related increase in the percentage of hindlimb bud cells in S-phase was evident at all dosages 4 hr after maternal dosing. By 28 hr postdosing, a normal cell cycle phase distribution was observed at doses of < 0.3 mg/kg. However, cell cycle perturbations persisted at higher dosages. NBS staining demonstrated increased cell death in areas of rapid cell division, indicative of replication-associated cytotoxicity, at doses of > or = 0.1 mg/kg. Observation of litters from additional dams killed at term revealed that at dosages of > or = 0.3 mg/kg, cleft palate and hindlimb defects were significantly elevated. In addition, above 0.3 mg/kg, fetal weight was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Quantitation of inhibition of DNA methylation of the retinoic acid receptor beta gene by 5-Aza-2'-deoxycytidine in tumor cells using a single-nucleotide primer extension assay.

The expression of several cancer-related genes has been reported to be silenced by DNA methylation of their promoter region. 5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent and specific inhibitor of DNA methylation, can reactivate the in vitro expression of these genes. In future clinical trials in tumor therapy with 5-AZA-CdR a method to quantitate its inhibition of methylation of specific tumor suppressor genes would provide important data for the analysis of the therapeutic efficacy of this analogue. We have modified the methylation-sensitive single-nucleotide primer extension assay reported by Gonzalgo and Jones (Nucleic Acids Res. 25, 2529-2531, 1997). Genomic DNA was treated with bisulfite and a fragment of the promoter region of the human retinoic acid receptor beta (RARbeta) gene, a tumor suppressor gene, was amplified using seminested PCR. Using two different primers we quantitated the inhibition of methylation produced by 5-AZA-CdR at two specific CpG sites in the RARbeta promoter in a human colon and a breast carcinoma cell line. The results obtained with the modified assay show a precise and reproducible quantitation of inhibition of DNA methylation produced by 5-AZA-CdR in tumor cells.

Antimetabolites, Antineoplastic↗
PubMed1995

HIV-1 expression induced by anti-cancer agents in latently HIV-1-infected ACH2 cells.

The expression of human immunodeficiency virus type 1 (HIV-1) in infected cells is induced (or enhanced) by a number of agents including phorbol myristate acetate (PMA), phytohemagglutinin (PHA), certain infectious agents, certain cytokines, and ultraviolet light. ACH2 cells represent latently HIV-1-infected T-cells, which produce only a low level of HIV-1 in vitro. We found that various anti-cancer agents including 5-azacytidine (5-AZC), 5-fluorouracil (5-FU), methotrexate, cytosine arabinoside, and vinblastine potentiated the expression of HIV-1 in ACH2 cells. There was no evidence of altered DNA methylation patterns in ACH2 cells cultured with 5-FU unlike with 5-AZC. The NF-kappa B binding activity was found to be enhanced in ACH2 cells exposed to 5-FU (but not in those exposed to 5-AZC) as assessed by the mobility shift assay using an oligonucleotide containing two NF-kappa B binding sites. These data suggest that the use of certain anti-cancer agents may induce (or enhance) the expression of HIV-1.

Antineoplastic Agents↗
PubMed1998

Analysis in Escherichia coli of the effects of in vivo CpG methylation catalyzed by the cloned murine maintenance methyltransferase.

Due in part to the complexity of mammalian systems, some of the proposed biological influences of mammalian DNA methylation have not been fully established. Escherichia coli cells, which normally contain negligible CpG methylation, exhibited progressive slowing of replication and lengthened generation times when expressing the murine DNA maintenance methyltransferase. Genomic analysis indicated significant amounts of CpG methylation in expressing cells which was absent from control cells. Expressing cells exposed to the cytosine demethylating agent, 5-azacytidine, rapidly reverted to propagation levels of controls. Substitution of cysteine with alanine in the carboxyl-terminal region proline-cysteine dipeptide of the methyltransferase completely inactivated methylating activity and cells expressing the inactive enzyme replicated as well as controls. These findings strongly implicate a role of epigenetic de novo CpG methylation in modulating cellular propagation, demonstrate that the maintenance methyltransferase can de novo methylate in vivo, and show that the methyltransferase requires an active site cysteine for activity.

Animals↗
PubMed1998

Involvement of DNA methylation in binding of a highly repetitive DNA component to nuclear scaffold proteins from rat liver.

Experimental reduction of the amount of CpG methylation in a highly repetitive DNA component was achieved by growth of Ac2F cells in the presence of 5-aza-2'-deoxycytidine or procainamide, as judged by the results of methyl-sensitive restriction endonuclease digestion and colony hybridization. Modification of genomic DNA with these DNA methylation inhibitors increased the release of 370-bp highly repetitive DNA from rat chromosomal DNA by HindIII digestion. This result indicated that highly repetitive DNA components in the nuclear scaffold fraction are hypermethylated. On the other hand, methylated DNA was used for southwestern analysis to investigate the protein(s) which bind specifically to the DNA in the nuclear scaffold fraction. The introduction of additional methylated cytosines within a highly repetitive DNA component affected the binding of DNA to the nuclear scaffold proteins. Thus, cytosine methylation may be involved in the regulation of gene expression and construction of the higher-order structure of chromatin.

Animals↗
PubMed1998

Increased expression of rat ribosomal protein L4 mRNA in 5-azacytidine-treated PC12 cells prior to apoptosis.

5-Azacytidine (5AzC), a cytidine analogue, is thought to induce apoptosis in fetal neuronal cells and PC12 cells through DNA hypomethylation. However, apoptosis can be inhibited by adding protein synthesis inhibitors, indicating de novo protein synthesis may be partially responsible for apoptosis. Therefore, genes expressed just before apoptosis from 5AzC-treated PC12 cells were cloned. cDNA libraries were prepared from both 5AzC-treated and untreated PC12 cells and these libraries were subtracted. One clone overexpressed in 5AzC-treated PC12 cells was obtained, and was identified as the nearly full length (9 nt at 5' end and 1 nt at 3' end missing) rat ribosomal protein L4 (rpL4) gene. Time course study of Northern blot analysis in 5AzC-treated PC12 cells revealed that the peak of rat rpL4 gene expression preceded DNA fragmentation. COS-7 cells transfected with different amounts of cDNA from the subtracted clone expressed rat rpL4 dose-dependently. DNA fragmentation in the transfected COS-7 cells occurred proportional to the amount of the cDNA used for transfection. The present study indicates that rat rpL4 gene expression selectively increases in PC12 cells prior to 5AzC-induced apoptosis and that COS-7 cells transfected with and expressing the rat rpL4 gene also undergo apoptosis.

Animals↗