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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 DNA synthesis in S phase synchronized HeLa cells. Left panel: images of the BrdUrd Comet assay performed at 2.5, 4, 6 and 7 hours from the beginning of DNA synthesis. The pictures, obtained by fluorescence microscopy show identical comet fields with FITC (green) and propidium iodide staining (red). C: control cells. T: treated cells. Right panel: Quantification of DNA synthesis at different time points from the beginning of S phase. (a): representative image of BrdUrd-Comet assay (INGR ×150, FITC staining), the percentage of tail DNA shared uniform distribution among the various comet formations. Tail moment measured following FITC (b) and PI (c) stainings, respectively. Controls (triangles); treated (squares). Results shown are the mean ± SE of 25 cells. (d): Thymidine incorporation per cell at 5 and 6 hours from the beginning of S phase. Control cells (black), treated cells (grey). The error bars show the standard deviation obtained from three independent experiments. Student’s test. *p < 0.05 **p < 0.01.
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Figure 1: Analysis of DNA synthesis in S phase synchronized HeLa cells. Left panel: images of the BrdUrd Comet assay performed at 2.5, 4, 6 and 7 hours from the beginning of DNA synthesis. The pictures, obtained by fluorescence microscopy show identical comet fields with FITC (green) and propidium iodide staining (red). C: control cells. T: treated cells. Right panel: Quantification of DNA synthesis at different time points from the beginning of S phase. (a): representative image of BrdUrd-Comet assay (INGR ×150, FITC staining), the percentage of tail DNA shared uniform distribution among the various comet formations. Tail moment measured following FITC (b) and PI (c) stainings, respectively. Controls (triangles); treated (squares). Results shown are the mean ± SE of 25 cells. (d): Thymidine incorporation per cell at 5 and 6 hours from the beginning of S phase. Control cells (black), treated cells (grey). The error bars show the standard deviation obtained from three independent experiments. Student’s test. *p < 0.05 **p < 0.01.

Mentions: Cell cycle arrest and apoptosis were induced in HeLa cells after incubation with a pool of peptides extracted from wheat bud chromatin [7]. A mechanism of action was proposed in which the inhibition of cell growth results from their ability to affect DNA replication. In order to provide a more detailed picture of their action on this process, we performed the BrdUrd Comet assay in synchronized HeLa cell cultures during the DNA synthesis. S phase cells were obtained by the double thymidine block that arrests the cells at the G1/S boundary. The removal of thymidine by replacement with normal medium induces the onset of S phase. DNA synthesis was analyzed by determination of 3H thymidine incorporation into DNA at 1 hour intervals. The time course of incorporation indicates that 3H thymidine uptake initiates shortly after the removal of the thymidine block, reaches the maximum after 6 hours and drops at 8 hours. This pattern demonstrates the occurrence of synchronization since cells not subjected to the thymidine block show a constant increasing rate of 3H thymidine incorporation (data not shown). Immediately after the removal of the thymidine block, the cells were incubated with the peptide pool for 2.5, 4, 6 and 7 hours, while replicating DNA was labelled by adding the thymidine analogue BrdUrd to the culture medium. The cells were then collected and subjected to the comet assay. The label incorporation was detected, following electrophoresis of the cells, by immunological assay. Figure 1 shows the localization of such label at different time points during DNA synthesis and the quantification of the DNA fragmentation (PI staining) and BrdUrd labelling (FITC staining) within the comet tails. In the control cells, at the beginning of the DNA synthesis (2.5 hours from the removal of thymidine block), little DNA fragmentation was detected. After 4 hours from the onset of the DNA synthesis, DNA fragmentation was detected and the BrdUrd label was localized close to the strand discontinuities, constituting the comet tails. In late DNA synthesis, 6 and 7 hours from the onset of the DNA replication, the level of the DNA strand breaks and BrdUrd incorporation in the tails decreased. In the treated cells, from the beginning (2.5 time point) till mid S phase, the levels of DNA fragmentation and tail labelling were significantly higher than in the controls. They reached the maximum value after 4 hours from the onset of the S phase, remained roughly the same at 6 hours and sharply decreased at the 7-hour time point. We determined the DNA synthesis activity at 5 and 6 hours from the beginning of the S phase by pulse labelling the cells with 3H thymidine for 30 min. The radioactivity incorporation per cell was lower in the treated cells respective to the control cells (Figure 1d). These data support our hypothesis of an action of this pool of peptides in the DNA replication process.


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 DNA synthesis in S phase synchronized HeLa cells. Left panel: images of the BrdUrd Comet assay performed at 2.5, 4, 6 and 7 hours from the beginning of DNA synthesis. The pictures, obtained by fluorescence microscopy show identical comet fields with FITC (green) and propidium iodide staining (red). C: control cells. T: treated cells. Right panel: Quantification of DNA synthesis at different time points from the beginning of S phase. (a): representative image of BrdUrd-Comet assay (INGR ×150, FITC staining), the percentage of tail DNA shared uniform distribution among the various comet formations. Tail moment measured following FITC (b) and PI (c) stainings, respectively. Controls (triangles); treated (squares). Results shown are the mean ± SE of 25 cells. (d): Thymidine incorporation per cell at 5 and 6 hours from the beginning of S phase. Control cells (black), treated cells (grey). The error bars show the standard deviation obtained from three 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 1: Analysis of DNA synthesis in S phase synchronized HeLa cells. Left panel: images of the BrdUrd Comet assay performed at 2.5, 4, 6 and 7 hours from the beginning of DNA synthesis. The pictures, obtained by fluorescence microscopy show identical comet fields with FITC (green) and propidium iodide staining (red). C: control cells. T: treated cells. Right panel: Quantification of DNA synthesis at different time points from the beginning of S phase. (a): representative image of BrdUrd-Comet assay (INGR ×150, FITC staining), the percentage of tail DNA shared uniform distribution among the various comet formations. Tail moment measured following FITC (b) and PI (c) stainings, respectively. Controls (triangles); treated (squares). Results shown are the mean ± SE of 25 cells. (d): Thymidine incorporation per cell at 5 and 6 hours from the beginning of S phase. Control cells (black), treated cells (grey). The error bars show the standard deviation obtained from three independent experiments. Student’s test. *p < 0.05 **p < 0.01.
Mentions: Cell cycle arrest and apoptosis were induced in HeLa cells after incubation with a pool of peptides extracted from wheat bud chromatin [7]. A mechanism of action was proposed in which the inhibition of cell growth results from their ability to affect DNA replication. In order to provide a more detailed picture of their action on this process, we performed the BrdUrd Comet assay in synchronized HeLa cell cultures during the DNA synthesis. S phase cells were obtained by the double thymidine block that arrests the cells at the G1/S boundary. The removal of thymidine by replacement with normal medium induces the onset of S phase. DNA synthesis was analyzed by determination of 3H thymidine incorporation into DNA at 1 hour intervals. The time course of incorporation indicates that 3H thymidine uptake initiates shortly after the removal of the thymidine block, reaches the maximum after 6 hours and drops at 8 hours. This pattern demonstrates the occurrence of synchronization since cells not subjected to the thymidine block show a constant increasing rate of 3H thymidine incorporation (data not shown). Immediately after the removal of the thymidine block, the cells were incubated with the peptide pool for 2.5, 4, 6 and 7 hours, while replicating DNA was labelled by adding the thymidine analogue BrdUrd to the culture medium. The cells were then collected and subjected to the comet assay. The label incorporation was detected, following electrophoresis of the cells, by immunological assay. Figure 1 shows the localization of such label at different time points during DNA synthesis and the quantification of the DNA fragmentation (PI staining) and BrdUrd labelling (FITC staining) within the comet tails. In the control cells, at the beginning of the DNA synthesis (2.5 hours from the removal of thymidine block), little DNA fragmentation was detected. After 4 hours from the onset of the DNA synthesis, DNA fragmentation was detected and the BrdUrd label was localized close to the strand discontinuities, constituting the comet tails. In late DNA synthesis, 6 and 7 hours from the onset of the DNA replication, the level of the DNA strand breaks and BrdUrd incorporation in the tails decreased. In the treated cells, from the beginning (2.5 time point) till mid S phase, the levels of DNA fragmentation and tail labelling were significantly higher than in the controls. They reached the maximum value after 4 hours from the onset of the S phase, remained roughly the same at 6 hours and sharply decreased at the 7-hour time point. We determined the DNA synthesis activity at 5 and 6 hours from the beginning of the S phase by pulse labelling the cells with 3H thymidine for 30 min. The radioactivity incorporation per cell was lower in the treated cells respective to the control cells (Figure 1d). These data support our hypothesis of an action of this pool of peptides in the DNA replication process.

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