RESUMO
BACKGROUND: We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis. METHODS: BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively. RESULTS: BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected. CONCLUSION: The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.
RESUMO
Dithiocarbamates are compounds commonly used in medicine and in agriculture and their prolonged use is known to result in neurotoxicity. Whether this response may be related to early gene expression has not been investigated. We have addressed this issue by mapping Fos expression in rats acutely injected with diethyldithiocarbamate (DDTC) and correlating these data to neural damage in the hippocampus as determined by pyknotic nuclei count. In comparison to saline injected rats, DDTC treatment induced a marked Fos expression in most brain regions at 1 and 3 h. In the hippocampus, a high Fos expression was followed by a variable number of pyknotic nuclei at 6 h, depending on the subregion. The data suggest that, in this model of neurotoxicity, c-fos induction does not reflect a cell commitment to die or survive, but rather a cell response to the DDTC-induced oxidative disorder.
