Gametocidal chromosomes enhancing chromosome aberration in common wheat induced by 5-azacytidine.

The gametocidal (Gc) chromosome from Aegilops spp induces chromosome mutation, which is introduced into common wheat as a tool of chromosome manipulation for genetic improvement. The Gc chromosome functions similar to a restriction-modification system in bacteria, in which DNA methylation is an important regulator. We treated root tips of wheat carrying Gc chromosomes with the hypomethylation agent 5-azacytidine; chromosome breakage and micronuclei were observed in these root tips. The frequency of aberrations differed in wheat containing different Gc chromosomes, suggesting different functions inducing chromosome breakage. Gc chromosome 3C caused the greatest degree of chromosome aberration, while Gc chromosome 3C(SAT) and 2C caused only slight chromosome aberration. Gc chromosome 3C induced different degrees of chromosome aberration in wheat varieties Triticum aestivum var. Chinese Spring and Norin 26, demonstrating an inhibition function in common wheat.

Satellite 2 demethylation induced by 5-azacytidine is associated with missegregation of chromosomes 1 and 16 in human somatic cells.

Satellite sequences are an important part of the pericentromeric regions in mammalian genomes; they play a relevant role in chromosome stability and DNA hypomethylation of these sequences has been reported in ICF syndrome and in some cancers that are closely associated with chromosomal abnormalities. Epigenetic modifications of satellite sequences and their consequences have not been extensively studied in human cells. In the present work, we evaluated satellite 2 methylation patterns in human lymphocytes exposed to 5-azacytidine (5-azaC) and assessed the relationship between these patterns and chromosome missegregation. Human lymphocytes were exposed to 10µM 5-azaC for 24, 48, and 72h. Segregation errors were evaluated in binucleate cells using FISH against pericentromeric regions of chromosomes 1, 9, and 16. DNA methylation patterns were evaluated by immunodetection, and by bisulfite plus urea conversion and sequencing. We have identified that 5-azaC induced missegregation of chromosomes 1 and 16, which have highly methylated satellite 2, after 72h of exposure. Chromosome methylation patterns showed a notable decrease in pericentromeric methylation. Bisulfite conversion and sequencing analysis demonstrated demethylation of satellite 2 associated to 5-azaC exposure, principally after 72h of treatment. This change occurred in a non-specific pattern. Our study demonstrates an association between loss of satellite 2 DNA methylation and chromosome loss in human lymphocytes.

Vitamin A and beta-carotene inhibitory effect during 1,2-dimethylhydrazine induced hepatocarcinogenesis potentiated by 5-azacytidine.

5-Azacytidine is being used for reactivation of tumor suppressor genes. However, its administration during DNA repair pontentiates hepatocarcinogenesis. We observed chemopreventive activities by vitamin A and beta-carotene during early hepatocarcinogenesis. Thus, in the present study we evaluated vitamin A and beta-carotene chemopreventive potential during early hepatocarcinogenesis potentiated by 5-azacytidine. Wistar rats received vitamin A (VAA group), beta-carotene (betaCA group) or corn oil (CO and COA groups). After three weeks of treatment, all animals were initiated with 1,2-dimethylhydrazine. Twelve hours later VAA, betaCA and COA groups received a single dose of 5-Azc. Hepatocytes were selected/promoted by 2-acetylaminofluorene and 70% partial hepatectomy. All animals were sacrificed six weeks after initiation. Compared to CO group (without 5-azacytidine), COA group presented higher (p<0.05) nodule multiplicity, larger (p<0.05) gamma-GT positive lesions that occupied a larger (p<0.05) area of liver section. Compared to COA group, VAA group presented decreased (p<0.05) nodule multiplicity while betaCA group tended to present smaller gamma-GT positive lesions and to decrease occupied liver section. These results reinforce vitamin A and beta-carotene chemopreventive potential. Considering that 5-azacytidine potentiates hepatocarcinogenesis, more studies are needed to elucidate the efficacy and safety of this drug for cancer control.

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.

Somatic recombination: a major genotoxic effect of two pyrimidine antimetabolitic chemotherapeutic drugs in Drosophila melanogaster.

Two deoxycytidine analogues, 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside, citarabine, araC) and 5-aza-2'-deoxycytidine (decitabine, DAC, 5-aza-dC), are the drugs of choice in the treatment of acute myeloid leukaemia. The araC-induced cytotoxicity is a direct result of its interference with nucleic acids synthesis, whereas 5-aza-dC is a potent suppressor of DNA methylation. We employed the standard version of the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster to evaluate the genotoxic potential of these two antimetabolites as a function of exposure concentration. In addition, we determined the relative contributions of mutational and recombinational events to total genotoxicity. The compounds were administered by chronic feeding of 3-day-old larvae. Our results indicate that recombinagenicity is the major genotoxic effect of araC and 5-aza-dC (approximately, 77 and 81%, respectively, recombination). The standardised clone induction frequencies (per mM concentration per cell per cell division) show that 5-aza-dC is 85 times more powerful then araC (inducing approximately 58 mutant clones per 10(5) cells per mM). The high recombinagenic activity of these two drugs suggests that--despite their therapeutic effects against cancer--a question is raised whether these drugs should be considered for adverse effects in cancer chemotherapy.