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A pool of peptides extracted from wheat bud chromatin inhibits tumor cell growth by causing defective DNA synthesis.

Mancinelli L, Secca T, De Angelis PM, Mancini F, Marchesini M, Marinelli C, Barberini L, Grignani F - Cell Div (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence of U2OS and HeLa cells treated with the peptide pool (2 μg/ml) for 24 and 96 hours. Top panel: confocal microscope images of phospho-foci stained with γH2AX antibody (green) and DAPI (blue). Lower panel: percentage of γH2AX positive cells, and average number of γH2AX foci per cell as scored from 100 cells. Error bars show the standard deviation obtained from four independent experiments. Student’s test. *p < 0.05 **p < 0.01.
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Figure 3: Immunofluorescence of U2OS and HeLa cells treated with the peptide pool (2 μg/ml) for 24 and 96 hours. Top panel: confocal microscope images of phospho-foci stained with γH2AX antibody (green) and DAPI (blue). Lower panel: percentage of γH2AX positive cells, and average number of γH2AX foci per cell as scored from 100 cells. Error bars show the standard deviation obtained from four independent experiments. Student’s test. *p < 0.05 **p < 0.01.

Mentions: In response to DNA replication stress, checkpoint proteins are activated to trigger the DNA repair machinery. A critical component of the signaling pathway is represented by the histone H2AX that, in its phosphorylated form γH2AX, specifically attracts proteins leading to the formation of nuclear DNA repair foci [8]. In order to investigate the induction of the DNA repair pathway following replication stress, we checked the expression of γH2AX in peptide treated cells. We tested two cell lines, HeLa and U2OS, that differ in their expression of the p53 protein. While in HeLa p53 is inactivated by the E6 protein encoded by the HPV genes [9], in U2OS it can be induced in the mediation of the cellular response to genotoxic stress. We first checked whether the peptide pool exerted antiproliferative effects also in U2OS cells with functional p53. We obtained (Figure 2) a level of growth inhibition in treated U2OS cultures comparable to that obtained for HeLa cells. In Figure 3 we show that the peptide treatment increases the percentage of γH2AX positive cells and the average number of γH2AX foci per cell in both cell lines. The involvement of p53 has been also investigated in U2OS cells by measuring the expression of phospho p53 (Ser-15) which is induced during the cellular response to DNA damage. The exposition of U2OS cells for 4 days to the peptide pool results in an increase of 4.5 times the control level of the expression of active p53. In agreement with Western Blot analysis, immunofluorescence results revealed a higher percentage of phospho-p53 (Ser-15) positive cells in the treated culture with a higher average level of fluorescence intensity per cell with respect to the control cells (Figure 4). Several small molecules with the ability to reactivate mutant p53 have been reported [10]. We therefore checked the possibility that in HeLa cells the treatment could interfere with the p53 degradation to recover its function, but no expression of phospho p53 was detected in this cell line (data not shown).


A pool of peptides extracted from wheat bud chromatin inhibits tumor cell growth by causing defective DNA synthesis.

Mancinelli L, Secca T, De Angelis PM, Mancini F, Marchesini M, Marinelli C, Barberini L, Grignani F - Cell Div (2013)

Immunofluorescence of U2OS and HeLa cells treated with the peptide pool (2 μg/ml) for 24 and 96 hours. Top panel: confocal microscope images of phospho-foci stained with γH2AX antibody (green) and DAPI (blue). Lower panel: percentage of γH2AX positive cells, and average number of γH2AX foci per cell as scored from 100 cells. Error bars show the standard deviation obtained from four independent experiments. Student’s test. *p < 0.05 **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750333&req=5

Figure 3: Immunofluorescence of U2OS and HeLa cells treated with the peptide pool (2 μg/ml) for 24 and 96 hours. Top panel: confocal microscope images of phospho-foci stained with γH2AX antibody (green) and DAPI (blue). Lower panel: percentage of γH2AX positive cells, and average number of γH2AX foci per cell as scored from 100 cells. Error bars show the standard deviation obtained from four independent experiments. Student’s test. *p < 0.05 **p < 0.01.
Mentions: In response to DNA replication stress, checkpoint proteins are activated to trigger the DNA repair machinery. A critical component of the signaling pathway is represented by the histone H2AX that, in its phosphorylated form γH2AX, specifically attracts proteins leading to the formation of nuclear DNA repair foci [8]. In order to investigate the induction of the DNA repair pathway following replication stress, we checked the expression of γH2AX in peptide treated cells. We tested two cell lines, HeLa and U2OS, that differ in their expression of the p53 protein. While in HeLa p53 is inactivated by the E6 protein encoded by the HPV genes [9], in U2OS it can be induced in the mediation of the cellular response to genotoxic stress. We first checked whether the peptide pool exerted antiproliferative effects also in U2OS cells with functional p53. We obtained (Figure 2) a level of growth inhibition in treated U2OS cultures comparable to that obtained for HeLa cells. In Figure 3 we show that the peptide treatment increases the percentage of γH2AX positive cells and the average number of γH2AX foci per cell in both cell lines. The involvement of p53 has been also investigated in U2OS cells by measuring the expression of phospho p53 (Ser-15) which is induced during the cellular response to DNA damage. The exposition of U2OS cells for 4 days to the peptide pool results in an increase of 4.5 times the control level of the expression of active p53. In agreement with Western Blot analysis, immunofluorescence results revealed a higher percentage of phospho-p53 (Ser-15) positive cells in the treated culture with a higher average level of fluorescence intensity per cell with respect to the control cells (Figure 4). Several small molecules with the ability to reactivate mutant p53 have been reported [10]. We therefore checked the possibility that in HeLa cells the treatment could interfere with the p53 degradation to recover its function, but no expression of phospho p53 was detected in this cell line (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus