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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

Analysis of p21 expression in Hela and U2OS cell lines. Western Blot analysis was performed in the cells treated for 24 and 96 hours with the peptide fraction. Densitometric quantification of p21 levels were obtained following normalization with tubulin. The molecular weight standards are shown on the left.
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Figure 5: Analysis of p21 expression in Hela and U2OS cell lines. Western Blot analysis was performed in the cells treated for 24 and 96 hours with the peptide fraction. Densitometric quantification of p21 levels were obtained following normalization with tubulin. The molecular weight standards are shown on the left.

Mentions: We also investigated the expression level of the p21 protein, an inhibitor of CDK-cyclin complexes, since it has been reported to arrest cell cycle progression in response to DNA damage. Western Blot analysis showed that the level of p21 expression in treated Hela and U2OS cells is higher compared to the controls (Figure 5). This suggests the involvement of p21 protein in the pathway adopted by the cells to induce G2 phase block that occurs after a defective DNA replication in the cells treated with the peptide pool.


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)

Analysis of p21 expression in Hela and U2OS cell lines. Western Blot analysis was performed in the cells treated for 24 and 96 hours with the peptide fraction. Densitometric quantification of p21 levels were obtained following normalization with tubulin. The molecular weight standards are shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Analysis of p21 expression in Hela and U2OS cell lines. Western Blot analysis was performed in the cells treated for 24 and 96 hours with the peptide fraction. Densitometric quantification of p21 levels were obtained following normalization with tubulin. The molecular weight standards are shown on the left.
Mentions: We also investigated the expression level of the p21 protein, an inhibitor of CDK-cyclin complexes, since it has been reported to arrest cell cycle progression in response to DNA damage. Western Blot analysis showed that the level of p21 expression in treated Hela and U2OS cells is higher compared to the controls (Figure 5). This suggests the involvement of p21 protein in the pathway adopted by the cells to induce G2 phase block that occurs after a defective DNA replication in the cells treated with the peptide pool.

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