Limits...
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

Flow cytometry analysis of the cells treated with the peptide pool. a: Percentage of cells in G1 (black), S (white) and G2 (grey) phases after 12 hours of peptide treatment (T) and after 24 hours of recovery (36-hour time point) in fresh medium (T rec.). C and C rec. represent the corresponding controls. The experiment shown is representative of three independent experiments. b: percentage of cells expressing CDK1cyclinB complex following 12 hours of treatment and 24 hours of recovery (36-hour time point) in normal medium. Control cell (black), treated cells (grey). 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
getmorefigures.php?uid=PMC3750333&req=5

Figure 6: Flow cytometry analysis of the cells treated with the peptide pool. a: Percentage of cells in G1 (black), S (white) and G2 (grey) phases after 12 hours of peptide treatment (T) and after 24 hours of recovery (36-hour time point) in fresh medium (T rec.). C and C rec. represent the corresponding controls. The experiment shown is representative of three independent experiments. b: percentage of cells expressing CDK1cyclinB complex following 12 hours of treatment and 24 hours of recovery (36-hour time point) in normal medium. Control cell (black), treated cells (grey). Error bars show the standard deviation obtained from three independent experiments. Student’s test. *p < 0.05 **p < 0.01.

Mentions: We have previously reported that cell growth inhibition is obtained when synchronized cells are treated during S phase only [7]. In order to assess the selectivity of the peptide action on the replication process, we evaluated the ability of the surviving cells to undergo a normal cell cycle progression after the removal of the peptide fraction from the culture medium. Unsynchronized HeLa cells were exposed to the peptide pool for 12 hours, then the pool was removed from the medium and cells were grown in normal medium for 24 hours. Cell cycle analysis and CDK1 expression level were measured at each time point. 12 hours of treatment induced G2 arrest and an accumulation of CDK1 kinase in the G2 cells. After 24 hours of recovery the cell distribution in the different phases of the cell cycle was the same as in the untreated cells. As shown in Figure 6, G2 arrest and CDK1 kinase accumulation were not detected. Taken together, these data demonstrate that the peptide pool exerts a selective action on S phase cells inducing a subsequent G2 arrest and apoptosis. The remaining cell population, unaffected, can continue cell cycle progression.


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)

Flow cytometry analysis of the cells treated with the peptide pool. a: Percentage of cells in G1 (black), S (white) and G2 (grey) phases after 12 hours of peptide treatment (T) and after 24 hours of recovery (36-hour time point) in fresh medium (T rec.). C and C rec. represent the corresponding controls. The experiment shown is representative of three independent experiments. b: percentage of cells expressing CDK1cyclinB complex following 12 hours of treatment and 24 hours of recovery (36-hour time point) in normal medium. Control cell (black), treated cells (grey). 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 6: Flow cytometry analysis of the cells treated with the peptide pool. a: Percentage of cells in G1 (black), S (white) and G2 (grey) phases after 12 hours of peptide treatment (T) and after 24 hours of recovery (36-hour time point) in fresh medium (T rec.). C and C rec. represent the corresponding controls. The experiment shown is representative of three independent experiments. b: percentage of cells expressing CDK1cyclinB complex following 12 hours of treatment and 24 hours of recovery (36-hour time point) in normal medium. Control cell (black), treated cells (grey). Error bars show the standard deviation obtained from three independent experiments. Student’s test. *p < 0.05 **p < 0.01.
Mentions: We have previously reported that cell growth inhibition is obtained when synchronized cells are treated during S phase only [7]. In order to assess the selectivity of the peptide action on the replication process, we evaluated the ability of the surviving cells to undergo a normal cell cycle progression after the removal of the peptide fraction from the culture medium. Unsynchronized HeLa cells were exposed to the peptide pool for 12 hours, then the pool was removed from the medium and cells were grown in normal medium for 24 hours. Cell cycle analysis and CDK1 expression level were measured at each time point. 12 hours of treatment induced G2 arrest and an accumulation of CDK1 kinase in the G2 cells. After 24 hours of recovery the cell distribution in the different phases of the cell cycle was the same as in the untreated cells. As shown in Figure 6, G2 arrest and CDK1 kinase accumulation were not detected. Taken together, these data demonstrate that the peptide pool exerts a selective action on S phase cells inducing a subsequent G2 arrest and apoptosis. The remaining cell population, unaffected, can continue cell cycle progression.

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